Methods for treating systemic lupus erythematosus with an anti-apolipoprotein B antibody

ABSTRACT

Described herein are peptides and antibodies for prevention and/or therapeutic treatment of mammals, including humans, against systemic lupus erythematosus, as well as diagnosing the presence or absence of antibodies related to increased or decreased risk of developing SLE and/or to disease grading, staging, and/or prognosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 120 as acontinuation-in-part of U.S. application Ser. No. 15/610,527, filed May31, 2017, now U.S. Pat. No. 10,434,141, which claims priority under 35U.S.C. § 119(e) to U.S. provisional patent application No. 62/343,601,filed May 31, 2016, the entireties of both of which are herebyincorporated by reference. This application also includes a claim ofpriority under 35 U.S.C. § 119(e) to U.S. provisional patent applicationNo. 62/678,940, filed May 31, 2018, the entirety of which is herebyincorporated by reference.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted May 31, 2019, as a text file named“SequenceListing-070017-000023US02_ST25” created on May 31, 2019 andhaving a size of 106,496 bytes, is hereby incorporated by reference.

TECHNICAL FIELD

The invention provides methods for treating systemic lupus erythematosus(SLE) and diagnosing SLE and cardiovascular disease in subject with SLE.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Accelerated atherosclerosis is a severe and life threateningcomplication of systemic lupus erythematosus (SLE). SLE is moreprevalent in women of childbearing age and African-Americans. Currenttreatment is non-specific immunosuppression with substantial seriousside effects. Even with these agents, patients remain at risk forcardiovascular complications. The incomplete response to treatmentindicates the need for more specific and less toxic therapies. It is nowunderstood that the initiation and progression of atherosclerosis in SLEpatients is modulated by the balance between opposing immune responses,atheroprotective and proatherogenic. Provided herein are methods fortreating SLE and diagnosing the likelihood of cardiovascular diseases insubject with SLE.

SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, compositions and methods whichare meant to be exemplary and illustrative, not limiting in scope.

Provided herein are methods for treating, inhibiting, preventing,reducing the severity of, slowing progression of, and/or promotingprophylaxis of a disease-state in a subject in need thereof. The methodsinclude providing a composition comprising one or more peptides ofApoB100 or derivatives, pharmaceutical equivalents, peptidomimetics oranalogs thereof; and administering an effective amount of thecomposition to the subject, so as to treat, inhibit, reduce the severityof, slow progression of and/or promote prophylaxis of the disease-statein the subject. In one embodiment, the disease-state is systemic lupuserythematosus (SLE). In another embodiment, the disease-state iscardiovascular disease. In a further embodiment, the disease-state iscardiovascular disease in the subject with SLE.

Also provided herein are methods for treating, inhibiting, preventing,reducing the severity of, slowing progression of, and/or promotingprophylaxis of a disease-state in a subject in need thereof. The methodsinclude providing a composition comprising CD8+ T cells activated withone or more peptides of ApoB100 or derivatives, pharmaceuticalequivalents, peptidomimetics or analogs thereof and (b) administering aneffective amount of the composition to the subject, so as to treat,inhibit, reduce the severity of, slow progression of and/or promoteprophylaxis of the disease-state in the subject. In one embodiment, thedisease-state is systemic lupus erythematosus (SLE). In anotherembodiment, the disease-state is cardiovascular disease. In a furtherembodiment, the disease-state is cardiovascular disease in the subjectwith SLE.

In various embodiments of the methods described herein, the peptides ofApoB100 are any one or more of peptides 1 to 302 of ApoB100 as set forthin SEQ ID NO: 1 to SEQ ID NO: 302. In one embodiment, the peptide ofApoB100 is P210 (SEQ ID NO: 210). In another embodiment, the peptide ofApoB100 is P45 (SEQ ID NO: 45). In some embodiments, the peptides ofApoB100 are fused to cholera toxin B (CTB). In one embodiment, P210 isfused to CTB. In another embodiment, P45 is fused to CTB.

Also provided herein are methods for diagnosing disease-states in asubject in need thereof. The methods include obtaining a sample from thesubject; assaying the sample to determine the level of autoantibodiesagainst ApoB100; and determining that the subject has increasedlikelihood of having the disease if the level of the autoantibodies isdecreased relative to a reference value, or determining that the subjecthas decreased likelihood of having the disease if the level ofautoantibodies is increased relative to a reference value. In oneembodiment, the disease-state is SLE. In another embodiment, thedisease-state is cardiovascular disease. In a further embodiment, thedisease-state is cardiovascular disease in subject with SLE.

Further provided herein are assays for determining the efficacy oftreatment for a disease in a subject in need thereof. The assayscomprise obtaining a sample from the subject; assaying the sample todetermine the level of autoantibodies against ApoB100; and determiningthat the treatment is effective if the level of the autoantibodies isincreased relative to a reference value, or determining that thetreatment is ineffective if the level of autoantibodies is decreasesrelative to a reference value. In one embodiment, the disease iscardiovascular disease in subject with SLE. In another embodiment, thedisease is SLE. In a further embodiment, the disease is cardiovasculardisease.

Also provided are methods for treating, reducing the severity orlikelihood of, slowing progression of or inhibiting systemic lupuserythematosus (SLE) in a subject, or atherosclerosis in a subjectexhibiting symptoms or having been diagnosed with SLE, which includesadministering to the subject an effective amount of an antibody orantibody fragment capable of binding to a fragment (comprising orconsisting of P45, SEQ ID NO: 45) of apolipoprotein B100 (ApoB100),wherein the antibody contains one, two or three heavy chaincomplementarity determining regions (HCDRs) selected from the groupconsisting of HCDR 1 (HCDR1), HCDR 2 (HCDR2) and HCDR 3 (HCDR3)sequences of SEQ ID Nos: 318, 319 and 320, respectively, and one, two orthree light chain complementarity determining regions (LCDRs) selectedfrom the group consisting of LCDR 1 (LCDR1), LCDR 2 (LCDR2) and LCDR 3(LCDR3) sequences of SEQ ID Nos: 321, 322 and 323, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts in accordance with various embodiments of the invention,body weight measurements for the duration of the study described inExample 1. Values=averages+/−SEM.

FIG. 2 depicts in accordance with various embodiments of the invention,food consumption measurements for the duration of the study.Values=averages+/−SEM.

FIG. 3 depicts in accordance with various embodiments of the invention,significant increase in oxPL with CVX-12 administration ingld.apoE^(−/−) mice on normal diet. Biotinylated ApoB (biot.ApoB),biotinylated antibody to oxidized phospholipids (biot.E06) and oxidizedphospholipid/apoB100 (OxPL/ApoB) in Example 1 for PBS, Alum, and CVX-12groups.

FIG. 4 depicts in accordance with various embodiments of the invention,anti-nuclear antibody reactivity detected in Example 1. Valuescorrespond to the maximum serum dilution at which ANA reactivity isdetectable, where 1=1:100, 2=1:1000, 4=1:10000, 6=1:30000, and8=1:90000.

FIG. 5 depicts in accordance with various embodiments of the invention,serum anti-cardiolipin antibody reactivity. Anti-cardiolipin antibodiesin serum of gld.apoE−/− mice on normal diet.

FIG. 6A-FIG. 6D depict in accordance with various embodiments of theinvention, atherosclerotic plaque coverage of en face aorta.Atherosclerotic lesion coverage is represented as a percentage of totalen face aortic area (plaque covered area/total area). Aorta plaquemeasurements are apoE−/− (FIG. 6A), gld (FIG. 6B), gld.apoE−/− on normaldiet (FIG. 6C), and gld.apoE−/− on western diet (FIG. 6D).

FIG. 7A-FIG. 7C depicts in accordance with various embodiments of theinvention described in example 1, atherosclerotic lesion area in aorticroots. Oil-red-O-stained cross-sections of aortic roots from apoE^(−/−)(FIG. 7A), gld.apoE^(−/−) normal diet (FIG. 7B), and gld.apoE^(−/−)western diet (FIG. 7C) mice. Representative images of each treatmentgroup (PBS, Alum, and CVX-12) and a measurement of cross-sectionalplaque area (mm²) are shown. P<0.05 for apoE^(−/−)-Alum (*) andapoE^(−/−)-CVX-12 (*) compared to apoE^(−/−)-PBS.

FIG. 8 depicts in accordance with various embodiments of the inventiondescribed in example 1, spleen and lymph node weights at sacrifice.Values=averages±SEM. P=0.017 for gld treated with CVX-12 versus gldtreated with PBS and P=0.002 for rosiglitazone versus normal dietgld.apoE−/− treated with PBS (*).

FIG. 9 depicts in accordance with various embodiments of the inventiondescribed in example 1, glomerular tuft size in lupus mouse models.Arrows designate glomerular tufts. Size of tuft area is measured bypixel count. SEM is shown.

FIG. 10A-FIG. 10C depict in accordance with various embodiments of theinvention, that death receptors ligands induces the release of solubledeath receptors in cell supernatant. The levels of the soluble deathreceptors (FIG. 10A) TRAILR2, (FIG. 10B) Fas and (FIG. 10C) TNFR1obtained from cell supernatant from freshly cultured healthy donor PBMCtreated with IL-1β (10 ng/ml), soluble Fas ligand (0.5 μg/ml), TNF (10ng/ml) or LPS (10 μg/ml).

FIG. 11A-FIG. 11F depicts in accordance with various embodiments of theinvention, that treatment of PBMC with soluble Fas ligand does notactivate apoptosis as extensively in SLE patients as in controls. Thelevels of the soluble death receptors (FIG. 11A) FAS (FIG. 11B) TRAILand (FIG. 11C) TNFR1. (FIG. 11D) Peripheral blood mononuclear cellsgated in the forward and site scatter lymphocyte gate shown as (FIG.11E) early apoptotic lymphocytic cells (Annexin⁺/7-AAD⁻) and (FIG. 11F)late apoptotic lymphocytic cells (Annexin⁺/7-AAD⁺) after treatment withsoluble Fas ligand (2.5 μg/ml).

FIG. 12 depicts in accordance with various embodiments of the invention,a flow chart showing the recruitment of the Malmo Diet and Cancercardiovascular cohort and the study participants of Example 3. See, *1.Manjer J, Elmståhl S, Janzon L, Berglund G. Invitation to apopulation-based cohort study: differences between subjects recruitedusing various strategies. Scand J Public Health. 2002; 30(2):103-112. 2.Manjer J, Carlsson S, Elmståhl S, Gullberg B, Janzon L, Lindström M,Mattisson I, Berglund G. The Malmö Diet and Cancer study:representativity, cancer incidence and mortality in participants andnon-participants. Eur J Cancer Prev. 2001 December; 10(6):489-499.

FIG. 13A-FIG. 13E depicts in accordance with various embodiments of theinvention, coronary event-free survival of autoantibody tertilesrecognizing the apolipoprotein B-100 (ApoB100) peptides P45 and P210during a 15-year follow-up. Kaplan-Meier event-free survival curves oftertiles of autoantibodies to ApoB100 peptides P45 and P210 revealed asignificant positive linear trend showing lower incidence of coronaryevents with increasing autoantibody levels. Tertiles of IgM-P45_(native)(FIG. 13A) IgM-P45_(MDA), (FIG. 13B) IgM-P210_(native), (FIG. 13C)IgM-P210_(MDA), (FIG. 13D) and IgG-P210_(native), (FIG. 13E) Log rank(Mantel Cox)] tests were used to calculate P for linear trends.

FIG. 14 depicts in accordance with various embodiments of the invention,one-dimensional box-plots showing median and 95% confidence interval forP45 immunoglobulin (Ig)M, malondialdehyde (MDA)-P45 IgM, P210 IgG andMDA-P210 IgG in systemic lupus erythematosus (SLE) patients with andwithout cardiovascular disease (CVD).

FIG. 15A-FIG. 15G depicts in accordance with various embodiments of theinvention, plaque composition and features in aortic arch sections ofMRL/lpr/Apoe^(−/−) mice after treatment with PBS, CVX-14 or Adjuphos.Oil Red O lipid staining fraction of aortic arch (FIG. 15A), plaque area(FIG. 15B), and CD68 fraction (FIG. 15C-FIG. 15D) in PBS, CVX-14 orAdjuphos treated groups. Carotid artery mRNA gene expression of FOXP3(FIG. 15E), TGF-β (FIG. 15F) and TNF-α (FIG. 15G).

FIG. 16A-FIG. 16E depict in accordance with various embodiments of theinvention, assessment of immune cells with flow cytometry aftertreatment of MRL/lpr/Apoe^(−/−) mice with PBS, CVX-14 or Adjuphos.Splenocytes amount of regulatory T cells (CD25+Foxp3+CD3+CD4+ cells,FIG. 16A), T helper cells (CD4+CD3+ cells, FIG. 16B), cytotoxic T cells(CD8+CD3+ cells, FIG. 16C), peripheral dendritic cells (B220+CD11c+,FIG. 16D) and monocytes (CD11b+CD115+, FIG. 16E) cell populations intreated mice.

FIG. 17 depicts, in accordance with an embodiment of the invention,atherosclerotic plaque development in the aortic arch ofMRL/lpr/Apoe^(−/−) mice. Lipid staining with Oil Red O after weeklyrepeatedly administered oral immunizations for ten weeks. Treatmentgroups PBS n=11, CTB 30 μg/ml n=13, P45-CTB 5 μg/ml n=13, P45-CTB 15μg/ml n=10 and P45-CTB 30 μg/ml n=12. Statistical analysis was performedusing One-way ANOVA with Sidak's multiple comparison test, comparing toPBS and CTB where **p<0.01. Each point represents one mouse.

FIG. 18A-FIG. 18I depict, in accordance with an embodiment of theinvention, splenocyte phenotype assessment with flow cytometry aftertreatment of MRL/lpr/Apoe^(−/−) mice with PBS, CTB 30 μg/ml, P45-CTB 5,15 and 30 μg/ml. Splenocyte percentage of (FIG. 18A) T helper cells(CD3⁺CD4⁺), (FIG. 18B) cytolytic T cells (CD3⁺CD8⁺), (FIG. 18C) T helpercentral memory cells (CD4⁺CD62L⁺CD44^(int-hi)), (FIG. 18D) T helpereffector cells (CD4⁺CD62⁻CD44^(hi)), (FIG. 18E) naïve T helper cells(CD4⁺CD62L⁺CD44^(lo)), (FIG. 18F) regulatory T cells(CD3⁺CD4⁺CD25⁺FoxP3⁺), (FIG. 18G) central memory cytolytic T cells(CD8⁺CD62L⁺CD44^(int-hi)), (FIG. 18H) cytolytic effector T cells(CD8⁺CD62⁻CD44^(hi)) and (FIG. 18I) naïve cytolytic Tcells)(CD8⁺CD62L⁺CD44^(lo). Statistical analysis was performed usingOne-way ANOVA with Sidak's multiple comparison test, comparing to PBSand CTB where *p<0.05, **p<0.01 and ***p<0.001. Each point representsone mouse.

FIG. 19A-FIG. 19I depicts, in accordance with an embodiment of theinvention, lymph node cell phenotype assessment with flow cytometryafter treatment of MRL/lpr/Apoe^(−/−) mice with PBS, CTB 30 μg/ml,P45-CTB 5, 15 and 30 μg/ml. Splenocyte percentage of (FIG. 19A) T helpercells (CD3⁺CD4⁺), (FIG. 19B) cytolytic T cells (CD3⁺CD8⁺), (FIG. 19C) Thelper central memory cells (CD4⁺CD62L⁺CD44^(int-hi)), (FIG. 19D) Thelper effector cells (CD4⁺CD62⁻CD44^(hi)), (FIG. 19E) naïve T helpercells (CD4⁺CD62L⁺CD44^(lo)), (FIG. 19F) regulatory T cells(CD3⁺CD4⁺CD25⁺FoxP3⁺), (FIG. 19G) central memory cytolytic T cells(CD8⁺CD62L⁺CD44^(int-hi)), (FIG. 19H) cytolytic effector T cells(CD8⁺CD62⁻CD44^(hi)) and (FIG. 19I) naïve cytolytic T cells(CD8⁺CD62L⁺CD44^(lo)). Statistical analysis was performed using One-wayANOVA with Sidak's multiple comparison test, comparing to PBS and CTBwhere *p<0.05, **p<0.01 and ***p<0.001. Each point represents one mouse.

FIG. 20 depicts, in accordance with an embodiment of the invention, flowcytometry analysis of blood from MRL/lpr/Apoe^(−/−) mice at treatmentday 42 of 56. Blood regulatory T cell (CD3+CD4+CD25+FoxP3+) percentagesduring treatment. Treatment groups PBS n=11, CTB 30 μg/ml n=13, P45-CTB5 μg/ml n=12, n=9 and P45-CTB 30 μg/ml n=11. Due to technicaldifficulties one mouse in P45-CTB 5 μg/ml, one mouse in P45-CTB 15 μg/mland two mice in P45-CTB 30 μg/ml groups were excluded.

FIG. 21A depicts the polynucleotide sequences encoding heavy chain (SEQID NO:303) and encoding light chain (SEQ ID NO: 304) of an exemplaryantibody (orticumab) for use with the methods described herein. FIG. 21Band the first ten lines of FIG. 21C depict the amino acid sequence ofthe heavy chain (SEQ ID NO: 316) of orticumab in upper case letters,which is translated from the adjacent lowercase letters showing thepolynucleotide sequence. Starting from line 15 of FIG. 21C, the aminoacid sequence of the light chain (SEQ ID NO: 317) of orticumab isdepicted in upper case letters, which is translated from the adjacentlowercase letters showing the polynucleotide sequence.

FIG. 22 depicts serum cholesterol and triglyceride levels at euthanasia.Values=averages±SEM. P<0.05 for cholesterol levels of gld.apoE−/− miceon western diet for PBS versus Alum and PBS versus CVX-12 (*). P<0.001for triglyceride levels gld.apoE−/− mice on rosiglitazone compared toPBS control mice (#).

FIG. 23 depicts anti-nuclear antibody (ANA) reactivity in mice fed withwestern diet (WD) detected in Example 1. Values correspond to themaximum serum dilution at which ANA reactivity is detectable, where1=1:100, 2=1:1000, 4=1:10000, 6=1:30000, and 8=1:90000.

FIG. 24 depicts T-regulatory cell populations from spleen. P<0.0001 forCD8+ cell populations in gld mice versus apoE−/− mice (*).

FIG. 25A depicts the plaque area (mm²) in SLE×ApoE^(−/−) mice.Progression of total plaque area (mm²) after passive immunization withbaseline, untreated, control IgG and human recombinant IgG against P45.Baseline, n=3; Untreated, n=6; Control antibody, n=10; BI-204(orticumab), n=6. Statistical analysis was performed using GraphPadPrism Software 5 with nonparametric two-tailed Mann-Whitney's t-testwhere *p<0.05. Quantitative data are presented as mean±S.E.M. FIG. 25Bdepicts the area of Oil-Red-O (ORO) staining (%) in SLE×ApoE^(−/−) miceinjected with a control antibody or with orticumab (BI-204).

FIG. 26 depicts the quantification and the staining micrographs ofCD68(%) in SLE×ApoE^(−/−) mice injected with a control antibody or withorticumab (BI-204).

FIG. 27 depicts the quantification and the staining micrographs of TUNELcells in SLE×ApoE^(−/−) mice injected with a control antibody or withorticumab (BI-204).

FIG. 28 depicts the quantifications of biomarkers in plasma inSLE×ApoE^(−/−) mice injected with a control antibody or with orticumab(BI-204).

FIG. 29 depicts the serum concentration of orticumab over time followingmultiple-dose intravenous administration of orticumab to human subjectsin a Phase I study.

FIG. 30 depicts the arithmetic mean serum concentration (linear scale)of orticumab over time (day) following a single dose of 360 mg orticumabin the second Phase I study.

FIG. 31 shows a schematic of the trial design of a Phase IIa study inExample 10.

FIG. 32 depicts a simulated human pharmacokinetics (PK) profile after asubcutaneous (SC) loading dose of 8 mg/kg followed by a weekly SC dosingof 2 mg/kg (the solid line). Dashed lines indicate two desiredthresholds (4 μg/mL, generally lowest line, and 12 μg/mL, the higherdashed line) of the serum concentration of orticumab.

FIG. 33A and FIG. 33B depict simulated human PK profiles based on SCdosing. In FIG. 33A, the highest solid line between 0 and 96 hoursindicates dosing at a weekly frequency at 2 mg/kg; generally the middlesolid line between 24 and 96 hours indicates dosing at a bi-weeklyfrequency at 2 mg/kg; and generally the lowest solid line indicatesdosing at a monthly frequency at 2 mg/kg. In FIG. 33B, the solid lineindicates a loading dose at 5 mg/kg and subsequent doses at 2 mg/kgevery two weeks. Generally lower dashed line indicates a desiredthreshold of 4 μg/mL of the serum concentration of orticumab. Generallyhigher dashed line indicates a desired threshold of 12 μg/mL of theserum concentration of orticumab.

FIG. 34 depicts the simulated human PK profiles after weekly SC dosing,using PK parameters from Phase I data. The solid lines in the graph fromlow to high represent dosages at 0.5, 1, 1.25, 2, 2.5, 4 or 6 mg/kg.

FIG. 35 depicts the simulated human PK profiles after bi-weekly SCdosing, using PK parameters from Phase I data. The solid lines in thegraph from low to high represent dosages at 1, 1.5, 2 or 2.5 mg/kg.

FIG. 36 depicts the simulated human PK profiles after monthly SC dosing,using parameters from Phase I data. The solid lines in the graph fromlow to high represent dosages at 2, 3, 4 or 6 mg/kg.

FIG. 37A-FIG. 37C depict response to oxLDL of PBMC from SLE patients andhealthy controls, characterized by plasma levels of oxLDL (37A), IL-6(37B) and MCP-1 (37C) in healthy controls (n=31) and SLE cases (n=53).

FIG. 38 depicts the gating strategy for CD3⁺, CD3⁻ and CD3⁻HLA-DR⁺populations from all viable (Zombie Aqua-) PBMC.

FIG. 39A-FIG. 39C depict CD3⁺ T cell populations in healthy controls(39A) and in SLE cases (39B) with or without exposure to 10 μg/ml ofoxLDL for 24 hours; FIG. 39C as a comparison.

FIG. 40A-FIG. 40C depict CD3⁻ HLA-DR⁺ antigen-presenting cellpopulations in healthy controls (40A) and in SLE cases (40B) with orwithout exposure to 10 μg/ml of oxLDL for 24 hours; FIG. 40 as acomparison.

FIG. 41A-FIG. 41C depict release of IL-6 in healthy controls (41A) andin SLE (41B) PBMC with or without exposure to 10 μg/ml of oxLDL for 24hours; FIG. 41C as a comparison. FIG. 42A-FIG. 42C depict release ofIFN-γ in healthy controls (42A) and in SLE (42B) PBMC with or withoutexposure to 10 μg/ml of oxLDL for 24 hours; FIG. 42C as a comparison.FIG. 43A-FIG. 43C depict release of IL-1RA in healthy controls (43A) andin SLE (43B) PBMC with or without exposure to 10 μg/ml of oxLDL for 24hours; FIG. 43C as a comparison. FIG. 44A-FIG. 44C depict release ofIL-10 in healthy controls (44A) and in SLE (44B) PBMC with or withoutexposure to 10 μg/ml of oxLDL for 24 hours; FIG. 44C as a comparison.Concentrations are normalized to the number of seeded cells. Each dotrepresents one individual patient in FIGS. 37A-37C and one samplereplicate. Cell population frequencies from each individual arepresented as percent and individual percent change. Wilcoxon pairedsamples rank test of same individual data in unstimulated vs oxLDLstimulated samples and Mann-Whitney rank test of individual plasma levelvalues and individual percent change were used, *p≤0.05, **p≤0.01 and***p≤0.001.

FIG. 45A and FIG. 45B depict that B6.lpr.ApoE−/− mice display a SLEphenotype, characterized by ApoE−/− and B6.lpr.ApoE−/− spleen cell count(45A), double negative T cells (CD4⁻CD8⁻ of CD3⁺) (45B) and anti-nuclearantibody presence from plasma diluted 1:100.

FIG. 46 depicts an experimental setup, where female B6.lpr.ApoE^(−/−)mice were fed high-fat diet (HFD) from 6 weeks of age until terminationof the study at 27 weeks of age. Mice were orally administered by gavagewith CTB (30 μg, n=13) or p45-CTB conjugate (5 μg, n=13) starting at 18weeks of age at day 0, 3, and 5 followed by once weekly administrationsuntil 26 weeks of age.

FIG. 47A-FIG. 47C depict the weight (47A), plasma triglyceride levels(47B) and plasma cholesterol levels (47C). FIG. 48A and FIG. 48B depictplasma P45-IgG and P45-IgM antibodies, respectively, at termination ofthe study. Each dot represents one mouse and data are shown asmean±standard deviation, *p≤0.05 with Students t-test on normallydistributed data or Mann-Whitney rank test on not normally distributeddata.

FIG. 49A and FIG. 49B demonstrate the mucosal immunization with p45-CTBis associated with a regulatory effect in draining mesenteric lymphnode, which is depicted by cell frequencies in mesenteric lymph node(49A) and in spleen (49B) of regulatory T cells (CD25⁺FoxP3⁺ gated fromCD3⁺CD4⁺). FIG. 50A and FIG. 50B depict cell frequencies in mesentericlymph node (50A) and in spleen (50B) of T effector memory cells(CD44⁺CD62L⁻ gated from CD3⁺CD4⁺). FIG. 51A and FIG. 51B depict cellfrequencies in mesenteric lymph node (51A) and in spleen (51B) offollicular B cells (CD21/35⁺CD23⁺ gated from B220 positive CD24⁻ cells).FIG. 52A and FIG. 52B depict cell frequencies in mesenteric lymph node(52A) and in spleen (52B) of regulatory B cells (CD1d^(hi)CD5⁺ gatedfrom B220⁺). Each dot represents one mouse and data is shown asmean±standard deviation, *p≤0.05 and ***p≤0.001 with Students t-test onnormally distributed data or Mann-Whitney rank test on not normallydistributed data.

FIG. 53-FIG. 56, FIG. 57A and FIG. 57B demonstrate p45-CTB treatmentreduced development of atherosclerotic plaques. Atherosclerosis in theaortic arch as assessed by en face percent Oil Red O lipid stained area(FIG. 53), by subvalvular plaque area (FIG. 54), by Oil Red O lipidstained area of subvalvular plaques (FIG. 55), by CD68 positive area ofsubvalvular plaques (FIG. 56), and by carotids artery mRNA expression ofFoxP3 (FIG. 57A) and IL-10 (FIG. 57B) at termination of the studyexpressed as log transformed relative fold change to CTB, normalized to18S as housekeeping gene. Gene expression data was log transformedbefore analysis. Each dot represents one mouse and data is shown asmean±standard deviation, *p≤0.05 and **p≤0.01 with Students t-test onnormally distributed data or Mann-Whitney rank test on not normallydistributed data.

FIG. 58-FIG. 61 demonstrate p45-CTB did not aggravate SLE phenotype.FIG. 58 depicts the score of plasma anti-nuclear antibody (ANA), whichis expressed as mean of four individual persons scoring independentlyaccording to a scoring matrix. FIG. 59 depicts the plasma anti-dsDNA.FIG. 60 depicts urine albumin:creatinine ratio (ACR). FIG. 61 depictsblood urea nitrogen concentrations. Four mice in CTB group were excludedin all urine analyses for technical reasons. Eight and three mice inP45-CTB were excluded from the albumin:creatinine ratio and blood ureanitrogen analyses respectively. Each dot represents one mouse and datais shown as mean±standard deviation, *p≤0.05 with Students t-test onnormally distributed data or Mann-Whitney rank test on not normallydistributed data.

FIG. 62, FIG. 63A, FIG. 63B, FIG. 64, FIG. 65A, FIG. 65B, FIG. 66A-FIG.66D, FIG. 67A and FIG. 67B demonstrate passive immunization with anApoB100 peptide antibody reduced atherosclerotic plaques development.FIG. 62 is a diagram depicting that male B6.lpr.ApoE^(−/−) mice were fedhigh-fat diet (HFD) at 6 weeks of age until termination of the study at22 weeks of age. Mice were given intra-peritoneal injections of controlIgG1 (CIgG, 1 mg, n=11) or MDA.p45-IgG1 (1 mg, n=7) at 18, 19 and 20weeks of age. FIG. 63A and FIG. 63B show Oil Red O lipid stained area ofen face aortic arch and subvalvular plaque area, respectively. FIG. 64depicts CD68 positive subvalvular plaque area including representativepictures of sections. FIG. 65A and FIG. 65B depict carotid artery mRNAexpression of IL-10 and MCP-1, respectively, at termination of the studyexpressed as log transformed relative fold change to CTB, normalized to18S housekeeping gene. Gene expression data was log transformed beforeanalysis. FIG. 66A-FIG. 66D depict the plasma concentrations of plasmatriglyceride (66A), plasma cholesterol (66B), plasma oxLDL (66C) andplasma IL-10 (66D) at termination of the study. Seven and six mice inCIgG group were excluded from gene expression analysis of IL-10 andMCP-1 respectively, two mice were excluded in MDA.P45-IgG group. FIG.67A and FIG. 67B depict glomerular area (67A) and cell count (67B) fromkidney. Five mice and one mouse in CIgG and MDA.p45-IgG respectivelywere excluded. Each dot represents one mouse and data is shown asmean±standard deviation, *p≤0.05 with Students t-test on normallydistributed data or Mann-Whitney rank test on not normally distributeddata.

FIG. 68A, FIG. 68B, FIG. 69-FIG. 72 and FIG. 73A-FIG. 73D demonstrateMDA.p45-IgG enhance uptake of oxLDL in CD14⁺ monocytes. OxLDL conjugatedto Dil-fluorochrome difference in mean MFI (68A) and in individual MFI(68B) from total SLE PBMC's (n=26). FIG. 69 depicts manually gated CD14+and DILoxLDL+ populations in concatenated data of all samples andvisualized in t-SNE plot of 24 h in vitro cultured PBMCs after oxLDL andoxLDL with MDA.P45-IgG respectively. Percent of CD14⁺ (FIG. 70) andCD14⁺ Dil-OxLDL⁺ double positive cells (FIG. 71) includingrepresentative plot from one individual from PBMCs exposed to oxLDL oroxLDL with MDA.P45-IgG (FIG. 72). Each dot represents one individual andin vitro stimulation. Cell population frequencies from each individualare presented as percent. Wilcoxon paired samples rank test of sameindividual data in oxLDL vs oxLDL with MDA.Pp45-IgG stimulated sampleswere used, *p≤0.05, **p≤0.01 and ***p≤0.001. FIG. 73A-FIG. 73D depictthe addition of the antibody (MDA.P45-IgG) had small effects on thecytokine release (IL-6, IFNγ and IL-10 in FIGS. 73A, 73B and 73C,respective) from SLE PBMC, except only a modest increase in theanti-inflammatory cytokine IL-1RA reaching statistical significance(FIG. 73D).

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Allen et al., Remington: The Science and Practice of Pharmacy22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al.,Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology 3^(rd) ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006);Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7^(th) ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton,Dictionary of DNA and Genome Technology 3^(rd) ed., Wiley-Blackwell(Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A LaboratoryManual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N.Y. 2012), provide one skilled in the art with a general guide to manyof the terms used in the present application. For references on how toprepare antibodies, see Greenfield, Antibodies A Laboratory Manual2^(nd) ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013);Köhler and Milstein, Derivation of specific antibody-producing tissueculture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul.6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No.5,585,089 (1996 December); and Riechmann et al., Reshaping humanantibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Other features and advantages of theinvention will become apparent from the following detailed description,taken in conjunction with the accompanying drawings, which illustrate,by way of example, various features of embodiments of the invention.Indeed, the present invention is in no way limited to the methods andmaterials described. For convenience, certain terms employed herein, inthe specification, examples and appended claims are collected here.

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areuseful to an embodiment, yet open to the inclusion of unspecifiedelements, whether useful or not. It will be understood by those withinthe art that, in general, terms used herein are generally intended as“open” terms (e.g., the term “including” should be interpreted as“including but not limited to,” the term “having” should be interpretedas “having at least,” the term “includes” should be interpreted as“includes but is not limited to,” etc.).

“Administering” and/or “administer” as used herein refer to any routefor delivering a pharmaceutical composition to a patient. Routes ofdelivery may include non-invasive peroral (through the mouth), topical(skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular andrectal) and inhalation routes, as well as parenteral routes, and othermethods known in the art. Parenteral refers to a route of delivery thatis generally associated with injection, including intraorbital,infusion, intraarterial, intracarotid, intracapsular, intracardiac,intradermal, intramuscular, intraperitoneal, intrapulmonary,intraspinal, intrasternal, intrathecal, intrauterine, intravenous,subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.Via the parenteral route, the compositions may be in the form ofsolutions or suspensions for infusion or for injection, or aslyophilized powders.

“Beneficial results” may include, but are in no way limited to,lessening or alleviating the severity of the disease condition,preventing the disease condition from worsening, curing the diseasecondition, preventing the disease condition from developing, loweringthe chances of a patient developing the disease condition and/orprolonging a patient's life or life expectancy. In some embodiments, thedisease condition is SLE.

The term “effective amount” as used herein refers to the amount of apharmaceutical composition comprising one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof, to decreaseat least one or more symptom of the disease or disorder, and relates toa sufficient amount of pharmacological composition to provide thedesired effect. The phrase “therapeutically effective amount” as usedherein means a sufficient amount of the composition to treat a disorder,at a reasonable benefit/risk ratio applicable to any medical treatment.

A therapeutically or prophylactically significant reduction in a symptomis, e.g. at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 125%, at least about 150% or more in a measured parameter ascompared to a control or non-treated subject or the state of the subjectprior to administering the peptides described herein. Measured ormeasurable parameters include clinically detectable markers of disease,for example, elevated or depressed levels of a biological marker, aswell as parameters related to a clinically accepted scale of symptoms ormarkers for atherosclerosis. It will be understood, however, that thetotal daily usage of the compositions and formulations as disclosedherein will be decided by the attending physician within the scope ofsound medical judgment. The exact amount required will vary depending onfactors such as the type of disease being treated, gender, age, andweight of the subject.

“Subject” or “individual” or “animal” or “patient” or “mammal,” is meantany subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include, but arenot limited to, humans, domestic animals, farm animals, zoo animals,sport animals, pet animals such as dogs, cats, guinea pigs, rabbits,rats, mice, horses, cattle, cows; primates such as apes, monkeys,orangutans, and chimpanzees; canids such as dogs and wolves; felids suchas cats, lions, and tigers; equids such as horses, donkeys, and zebras;food animals such as cows, pigs, and sheep; ungulates such as deer andgiraffes; rodents such as mice, rats, hamsters and guinea pigs; and soon. In certain embodiments, the mammal is a human subject.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition,prevent the pathologic condition, pursue or obtain beneficial results,or lower the chances of the individual developing the condition even ifthe treatment is ultimately unsuccessful. Those in need of treatmentinclude those already with the condition as well as those prone to havethe condition or those in whom the condition is to be prevented.

“Patient outcome” refers to whether a patient survives or dies as aresult of treatment. A more accurate prognosis for patients as providedin this invention increases the chances of patient survival.

“Poor Prognosis” means that the prospect of survival and recovery ofdisease is unlikely despite the standard of care for the treatment ofthe cancer (for example, lung cancer), that is, surgery, radiation,chemotherapy. Poor prognosis is the category of patients whose survivalis less than that of the median survival.

“Good Prognosis” means that the prospect of survival and recovery ofdisease is likely with the standard of care for the treatment of thedisease, for example, surgery, radiation, chemotherapy. Good prognosisis the category of patients whose survival is not less than that of themedian survival.

“Pharmaceutically acceptable carriers” as used herein refer toconventional pharmaceutically acceptable carriers useful in thisinvention.

The term “fusion protein” as used herein indicates a protein createdthrough the attaching of two or more polypeptides which originated fromseparate proteins. In particular fusion proteins can be created byrecombinant DNA technology and are typically used in biological researchor therapeutics. Fusion proteins can also be created through chemicalcovalent conjugation with or without a linker between the polypeptidesportion of the fusion proteins.

The term “attach” or “attached” as used herein, refers to connecting oruniting by a bond, link, force or tie in order to keep two or morecomponents together, which encompasses either direct or indirectattachment such that for example where a first polypeptide is directlybound to a second polypeptide or material, and the embodiments whereinone or more intermediate compounds, and in particular polypeptides, aredisposed between the first polypeptide and the second polypeptide ormaterial.

The term “protein” or “polypeptide” as used herein indicates an organicpolymer composed of two or more amino acid monomers and/or analogsthereof. The term “polypeptide” includes amino acid polymers of anylength including full length proteins and peptides, as well as analogsand fragments thereof. A polypeptide of three or more amino acids isalso called an oligopeptide. As used herein the term “amino acid”,“amino acidic monomer”, or “amino acid residue” refers to any of thetwenty naturally occurring amino acids including synthetic amino acidswith unnatural side chains and including both D and L optical isomers.The term “amino acid analog” refers to an amino acid in which one ormore individual atoms have been replaced, either with a different atom,isotope, or with a different functional group but is otherwise identicalto its natural amino acid analog

“Peptidomimetic” as used herein is a small protein-like chain designedto mimic a protein function. They may be modifications of an existingpeptide or newly designed to mimic known peptides. They may be, forexample peptoids and/or β-peptides and/or D-peptides.

“Recombinant virus” refers to a virus that has been genetically altered(e.g., by the addition or insertion of a heterologous nucleic acidconstruct into the particle).

A “gene” or “coding sequence” or a sequence which “encodes” a particularprotein or peptide is a nucleic acid molecule that is transcribed (inthe case of DNA) and translated (in the case of mRNA) into a polypeptidein vitro or in vivo when placed under the control of appropriateregulatory sequences. The boundaries of the gene are determined by astart codon at the 5′ (i.e., amino) terminus and a translation stopcodon at the 3′ (i.e., carboxy) terminus. A gene can include, but is notlimited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNAsequences from prokaryotic or eukaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the gene sequence.

The term “control elements” refers collectively to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which collectively provide forthe replication, transcription and translation of a coding sequence in arecipient cell. Not all of these control elements need always bepresent, so long as the selected coding sequence is capable of beingreplicated, transcribed and translated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region including a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting foreign DNA into host cells. Such methods can resultin transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene transfer provides a unique approachfor the treatment of acquired and inherited diseases. A number ofsystems have been developed for gene transfer into mammalian cells. See,e.g., U.S. Pat. No. 5,399,346. Examples of well-known vehicles for genetransfer include adenovirus and recombinant adenovirus (RAd),adeno-associated virus (AAV), herpes simplex virus type 1 (HSV-1), andlentivirus (LV).

“Genetically modified cells”, “genetically engineered cells”, or“modified cells” as used herein refer to cells that express thepolynucleotide having the sequence of any one or more SEQ ID Nos: 1-302or combinations thereof or variants, derivatives, pharmaceuticalequivalents, peptidomimetics or analogs thereof. In one embodiment, thecell express the polynucleotide having the sequence of SEQ ID NO: 210 ora variant, derivative, pharmaceutical equivalent, peptidomimetic or ananalog thereof.

“Naked DNA” as used herein refers to DNA encoding a polypeptide havingthe sequence of any one or more of SEQ ID Nos: 1-302 or a combinationthereof, or a variant, derivative, pharmaceutical equivalent,peptidomimetic or an analog thereof, cloned in a suitable expressionvector in proper orientation for expression. Viral vectors which may beused include but are not limited SIN lentiviral vectors, retroviralvectors, foamy virus vectors, adeno-associated virus (AAV) vectors,hybrid vectors and/or plasmid transposons (for example sleeping beautytransposon system) or integrase based vector systems. Other vectors thatmay be used in connection with alternate embodiments of the inventionwill be apparent to those of skill in the art.

“Polynucleotide” as used herein includes but is not limited to DNA, RNA,cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA),shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (shortnucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, syntheticRNA, and/or tRNA.

The term “transfection” is used herein to refer to the uptake of foreignDNA by a cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. Virology,52:456 (1973); Sambrook et al. Molecular Cloning, a laboratory manual,Cold Spring Harbor Laboratories, New York (1989); Davis et al., BasicMethods in Molecular Biology, Elsevier (1986), and Chu et al. Gene13:197 (1981). Such techniques can be used to introduce one or moreexogenous DNA moieties, such as a plasmid vector and other nucleic acidmolecules, into suitable host cells. The term refers to both stable andtransient uptake of the genetic material.

“Vector”, “cloning vector” and “expression vector” as used herein referto the vehicle by which a polynucleotide sequence (e.g. a foreign gene)can be introduced into a host cell, so as to transform the host andpromote expression (e.g. transcription and translation) of theintroduced sequence. Vectors include plasmids, phages, viruses, etc.

Therapeutic Methods

Provided herein are methods for treating, inhibiting, preventing,reducing the severity of, slowing progression of and/or promotingprophylaxis of SLE in a subject in need thereof. The methods includeproviding one or more peptides of ApoB100 or derivatives, pharmaceuticalequivalents, peptidomimetics or analogs thereof and administering atherapeutically or prophylactically effective amount of the one or morepeptides to the subject so as to treat, inhibit, reduce the severity ofand/or promote prophylaxis of SLE in the subject. In some embodiments,the subject with SLE is diagnosed with or is suspected of havingcardiovascular disease. In some embodiments, the one or more peptides ofApoB100 are peptides having sequences set forth in SEQ ID Nos: 1-302 oras described in Table 1. In some embodiments, the one or more peptidesof ApoB100 are immunogenic fragments of the peptides set forth in SEQ IDNos 1-302 or as described in Table 1. In some embodiments, the ApoB100peptides for uses in the therapeutic methods described herein areadministered sequentially or simultaneously with antibodies specific toApoB100. In exemplary embodiments, the antibodies comprise, consist ofor consist essentially of the sequence set forth in SEQ ID NOs: 303and/or 304. In some embodiments, the peptides described herein areadministered sequentially or simultaneously with existing treatments forSLE. In some embodiments, the antibodies described herein areadministered sequentially or simultaneously with existing treatments forSLE. In some embodiments, the peptides and antibodies described hereinare administered sequentially or simultaneously with existing treatmentsfor SLE. In some embodiments, the one or more peptides of ApoB100 areadministrated to the subject 1-3 times per day or 1-7 times per week. Insome embodiments, the one or more peptides of ApoB100 are administratedto the subject for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years. In oneembodiment, the ApoB100 peptide for use in the therapeutic methodsdescribed herein is the peptide P210 having the sequence set forth inSEQ ID NO: 210, or a derivative, pharmaceutical equivalent,peptidomimetic or analog thereof. In one embodiment, the ApoB100 peptidefor use in the therapeutic methods described herein is the peptide P45having the sequence set forth in SEQ ID NO: 45, or a derivative,pharmaceutical equivalent, peptidomimetic or analog thereof. In someembodiments, the ApoB100 peptide for use in the therapeutic methodsdescribed herein are fused to CTB. In one embodiment, the ApoB100peptide for use in the therapeutic methods described herein is thepeptide P210 having the sequence set forth in SEQ ID NO: 210, or aderivative, pharmaceutical equivalent, peptidomimetic or analog thereofand is fused to CTB (P210-CTB). In one embodiment, the ApoB100 peptidefor use in the therapeutic methods described herein is the peptide P45having the sequence set forth in SEQ ID NO: 45, or a derivative,pharmaceutical equivalent, peptidomimetic or analog thereof and is fusedto CTB (P45-CTB).

Also provided herein are methods for treating, inhibiting, preventing,reducing the severity of, slowing progression of and/or promotingprophylaxis of cardiovascular diseases in a subject in need thereof. Themethods include providing one or more peptides of ApoB100 orderivatives, pharmaceutical equivalents, peptidomimetics or analogsthereof and administering a therapeutically or prophylacticallyeffective amount of the one or more peptides to the subject so as totreat, inhibit, reduce the severity of and/or promote prophylaxis ofcardiovascular disease in the subject. In some embodiments, the subjectwith cardiovascular disease is diagnosed with or is suspected of havingSLE. In some embodiments, the one or more peptides of ApoB100 arepeptides having sequences set forth in SEQ ID Nos: 1-302 or as describedin Table 1. In some embodiments, the one or more peptides of ApoB100 areimmunogenic fragments of the peptides set forth in SEQ ID Nos 1-302 oras described in Table 1. In some embodiments, the ApoB100 peptides foruses in the therapeutic methods described herein are administeredsequentially or simultaneously with antibodies specific to ApoB100. Inexemplary embodiments, the antibodies comprise the sequence set forth inSEQ ID NOs: 303 and/or 304. In some embodiments, the peptides describedherein are administered sequentially or simultaneously with treatmentsfor SLE. In some embodiments, the antibodies described herein areadministered sequentially or simultaneously with treatments for SLE. Insome embodiments, the peptides and antibodies described herein areadministered sequentially or simultaneously with treatments for SLE. Invarious embodiments, the ApoB100 peptides described herein orderivatives, pharmaceutical equivalents, peptidomimetics or analogsthereof are administrated to the subject before, during, or after thesubject having or suspected of having SLE develops the cardiovasculardisease. In some embodiments, the one or more peptides of ApoB100 areadministrated to the subject 1-3 times per day or 1-7 times per week. Insome embodiments, the one or more peptides of ApoB100 are administratedto the subject for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years. In oneembodiment, the ApoB100 peptide for use in the therapeutic methodsdescribed herein is the peptide P210 having the sequence set forth inSEQ ID NO: 210, or a derivative, pharmaceutical equivalent,peptidomimetic or analog thereof. In some embodiments, the methodsdescribed herein further comprise co-administering, eithersimultaneously or sequentially, existing therapies for atherosclerosiswith the peptides described herein. In one embodiment, the ApoB100peptide for use in the therapeutic methods described herein is thepeptide P45 having the sequence set forth in SEQ ID NO: 45, or aderivative, pharmaceutical equivalent, peptidomimetic or analog thereof.In some embodiments, the ApoB100 peptide for use in the therapeuticmethods described herein are fused to CTB. In one embodiment, theApoB100 peptide for use in the therapeutic methods described herein isthe peptide P210 having the sequence set forth in SEQ ID NO: 210, or aderivative, pharmaceutical equivalent, peptidomimetic or analog thereofand is fused to CTB (P210-CTB). In one embodiment, the ApoB100 peptidefor use in the therapeutic methods described herein is the peptide P45having the sequence set forth in SEQ ID NO: 45, or a derivative,pharmaceutical equivalent, peptidomimetic or analog thereof and is fusedto CTB (P45-CTB).

Also provided herein are methods for treating, inhibiting, preventing,reducing the severity of, slowing progression of and/or promotingprophylaxis of cardiovascular diseases in subjects having SLE orsuspected of having SLE. The methods include providing one or morepeptides of ApoB100 or derivatives, pharmaceutical equivalents,peptidomimetics or analogs thereof and administering a therapeuticallyor prophylactically effective amount of the one or more peptides to thesubject so as to treat, inhibit, reduce the severity of and/or promoteprophylaxis of SLE in the subject. In some embodiments, the one or morepeptides of ApoB100 are peptides having sequences set forth in SEQ IDNos: 1-302 or as described in Table 1. In some embodiments, the one ormore peptides of ApoB100 are immunogenic fragments of the peptides setforth in SEQ ID Nos 1-302 or as described in Table 1. In someembodiments, the ApoB100 peptides for uses in the therapeutic methodsdescribed herein are administered sequentially or simultaneously withantibodies specific to ApoB100. In exemplary embodiments, the antibodiescomprise the sequence set forth in SEQ ID NOs: 303 and/or 304. In someembodiments, the peptides described herein are administered sequentiallyor simultaneously with existing treatments for SLE. In some embodiments,the antibodies described herein are administered sequentially orsimultaneously with existing treatments for SLE. In some embodiments,the peptides and antibodies described herein are administeredsequentially or simultaneously with existing treatments for SLE. In someembodiments, the one or more peptides of ApoB100 are administrated tothe subject 1-3 times per day or 1-7 times per week. In someembodiments, the one or more peptides of ApoB100 are administrated tothe subject for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years. In oneembodiment, the ApoB100 peptide for use in the therapeutic methodsdescribed herein is the peptide P210 having the sequence set forth inSEQ ID NO: 210, or a derivative, pharmaceutical equivalent,peptidomimetic or analog thereof. In one embodiment, the ApoB100 peptidefor use in the therapeutic methods described herein is the peptide P45having the sequence set forth in SEQ ID NO: 45, or a derivative,pharmaceutical equivalent, peptidomimetic or analog thereof. In someembodiments, the ApoB100 peptide for use in the therapeutic methodsdescribed herein are fused to CTB. In one embodiment, the ApoB100peptide for use in the therapeutic methods described herein is thepeptide P210 having the sequence set forth in SEQ ID NO: 210, or aderivative, pharmaceutical equivalent, peptidomimetic or analog thereofand is fused to CTB (P210-CTB). In one embodiment, the ApoB100 peptidefor use in the therapeutic methods described herein is the peptide P45having the sequence set forth in SEQ ID NO: 45, or a derivative,pharmaceutical equivalent, peptidomimetic or analog thereof and is fusedto CTB (P45-CTB).

Further provided herein are methods for treating, inhibiting,preventing, reducing the severity of, slowing progression of and/orpromoting prophylaxis of SLE in a subject in need thereof. The methodsinclude providing CD8+ T cells activated with one or more peptides ofApoB100 or derivatives, pharmaceutical equivalents, peptidomimetics oranalogs thereof and administering a therapeutically or prophylacticallyeffective amount of the one or more peptides to the subject so as totreat, inhibit, reduce the severity of and/or promote prophylaxis of SLEin the subject. In some embodiments, the subject with SLE has or issuspected of having cardiovascular disease. In some embodiments, the oneor more peptides of ApoB100 are peptides having sequences set forth inSEQ ID Nos: 1-302 or as described in Table 1. In some embodiments, theone or more peptides of ApoB100 are immunogenic fragments of thepeptides set forth in SEQ ID Nos 1-302 or as described in Table 1. Insome embodiments, the CD8+ T cells activated with the ApoB100 peptidesfor uses in the therapeutic methods described herein are administeredsequentially or simultaneously with antibodies specific to ApoB100. Inexemplary embodiments, the antibodies comprise the sequence set forth inSEQ ID NOs: 303 and/or 304. In some embodiments, the CD8+ T cellsactivated with the ApoB100 peptides described herein are administeredsequentially or simultaneously with existing treatments for SLE. In someembodiments, the antibodies described herein are administeredsequentially or simultaneously with existing treatments for SLE. In someembodiments, the CD8+ T cells activated with the ApoB100 peptides andthe antibodies described herein are administered sequentially orsimultaneously with existing treatments for SLE. In some embodiments,the CD8+ T cells activated with the ApoB100 peptides or derivatives,pharmaceutical equivalents, peptidomimetics or analogs thereof areadministrated to the subject 1-3 times per day or 1-7 times per week. Insome embodiments, the CD8+ T cells activated with the ApoB100 peptidesor derivatives, pharmaceutical equivalents, peptidomimetics or analogsthereof are administrated to the subject for 1-5 days, 1-5 weeks, 1-5months, or 1-5 years. In one embodiment, the CD8+ T cells are activatedwith the peptide P210 having the sequence set forth in SEQ ID NO: 210,or a derivative, pharmaceutical equivalent, peptidomimetic or analogthereof. In one embodiment, the CD8+ T cells are activated with thepeptide P45 having the sequence set forth in SEQ ID NO: 45, or aderivative, pharmaceutical equivalent, peptidomimetic or analog thereof.In one embodiment, the CD8+ T cells are activated with the peptide P45having the sequence set forth in SEQ ID NO: 45, or a derivative,pharmaceutical equivalent, peptidomimetic or analog thereof. In someembodiments, the methods described herein further compriseco-administering, either simultaneously or sequentially, existingtherapies for SLE with the CD8+ T cells activated with the ApoB100peptides as described herein.

Also provided herein are methods for treating, inhibiting, preventing,reducing the severity of, slowing progression of and/or promotingprophylaxis of cardiovascular diseases in a subject in need thereof. Themethods include providing CD8+ T cells activated with one or morepeptides of ApoB100 or derivatives, pharmaceutical equivalents,peptidomimetics or analogs thereof and administering a therapeuticallyor prophylactically effective amount of the one or more peptides to thesubject so as to treat, inhibit, reduce the severity of and/or promoteprophylaxis of cardiovascular disease in the subject. In someembodiments, the subject with cardiovascular disease is diagnosed withor is suspected of having SLE. In some embodiments, the one or morepeptides of ApoB100 are peptides having sequences set forth in SEQ IDNos: 1-302 or as described in Table 1. In some embodiments, the one ormore peptides of ApoB100 are immunogenic fragments of the peptides setforth in SEQ ID Nos 1-302 or as described in Table 1. In someembodiments, the CD8+ T cells activated with the ApoB100 peptides foruses in the therapeutic methods described herein are administeredsequentially or simultaneously with antibodies specific to ApoB100. Inexemplary embodiments, the antibodies comprise the sequence set forth inSEQ ID NOs: 303 and/or 304. In some embodiments, the CD8+ T cellsactivated with the ApoB100 peptides described herein are administeredsequentially or simultaneously with treatments for SLE. In someembodiments, the antibodies described herein are administeredsequentially or simultaneously with treatments for SLE. In someembodiments, the CD8+ T cells activated with the ApoB100 peptides andthe antibodies described herein are administered sequentially orsimultaneously with treatments for SLE. In various embodiments, the CD8+T cells activated with the ApoB100 peptides or derivatives,pharmaceutical equivalents, peptidomimetics or analogs thereof, areadministrated to the subject before, during, or after the subject havingor suspected of having SLE develops the cardiovascular disease. In someembodiments, the CD8+ T cells activated with the ApoB100 peptides orderivatives, pharmaceutical equivalents, peptidomimetics or analogsthereof are administrated to the subject 1-3 times per day or 1-7 timesper week. In some embodiments, the CD8+ T cells activated with theApoB100 peptides or derivatives, pharmaceutical equivalents,peptidomimetics or analogs thereof are administrated to the subject for1-5 days, 1-5 weeks, 1-5 months, or 1-5 years. In one embodiment, theCD8+ T cells are activated with the peptide P210 having the sequence setforth in SEQ ID NO: 210, or a derivative, pharmaceutical equivalent,peptidomimetic or analog thereof. In some embodiments, the methodsdescribed herein further comprise co-administering, eithersimultaneously or sequentially, existing therapies for atherosclerosiswith the peptides described herein. In one embodiment, the CD8+ T cellsare activated with the peptide P45 having the sequence set forth in SEQID NO: 45, or a derivative, pharmaceutical equivalent, peptidomimetic oranalog thereof.

As described herein, in some embodiments, the peptides and/or antibodiesdescribed herein are administered sequentially or simultaneously withtreatments for SLE. In exemplary embodiments, existing treatments forSLE include but are not limited to systemic inflammation directedtreatments (such as antimalarials-hydroxychloroquine, corticosteroids,cyclophosphamide, mycophenolate mofetil, azathioprine, methotrexate orcombinations thereof), immune cell-targeted therapies (such as anti-CD20(rituximab), anti-CD22 (epratuzumab), abetimus (LJP-394), belimumab,atacicept), agents that target co-stimulatory pathways, anti-cytokinetherapies, memantine, intravenous immunoglobulin (IVIG), DNAvaccinations, or combinations thereof. In exemplary embodiments,existing treatments for atherosclerosis include but are not limited tostatins, anti-platelet agents, beta blockers, angiotensin-convertingenzyme (ACE) inhibitors, calcium channel blockers, surgery (such asangioplasty and stent placement, fibrinolytic therapy, percutaneouscoronary intervention (PCI), coronary artery bypass grafting (CABG),carotid endarterectomy), or combinations thereof.

In various embodiments, the therapeutically or prophylacticallyeffective amount of any one or more of the ApoB100 peptides and/or acombinations thereof, or analogs, pharmaceutical equivalents or apeptidomimetics thereof for use with the methods described herein is anyone or more of about 0.01 to 0.05 μg/kg/day, 0.05-0.1 μg/kg/day, 0.1 to0.5 μg/kg/day, 0.5 to 5 μg/kg/day, 0.5 to 1 μg/kg/day, 1 to 5 μg/kg/day,5 to 10 μg/kg/day, 10 to 20 μg/kg/day, 20 to 50 μg/kg/day, 50 to 100μg/kg/day, 100 to 150 μg/kg/day, 150 to 200 μg/kg/day, 200 to 250μg/kg/day, 250 to 300 μg/kg/day, 300 to 350 μg/kg/day, 350 to 400μg/kg/day, 400 to 500 μg/kg/day, 500 to 600 μg/kg/day, 600 to 700μg/kg/day, 700 to 800 μg/kg/day, 800 to 900 μg/kg/day, 900 to 1000μg/kg/day, 0.01 to 0.05 mg/kg/day, 0.05-0.1 mg/kg/day, 0.1 to 0.5mg/kg/day, 0.5 to 1 mg/kg/day, 1 to 5 mg/kg/day, 5 to 10 mg/kg/day, 10to 15 mg/kg/day, 15 to 20 mg/kg/day, 20 to 50 mg/kg/day, 50 to 100mg/kg/day, 100 to 200 mg/kg/day, 200 to 300 mg/kg/day, 300 to 400mg/kg/day, 400 to 500 mg/kg/day, 500 to 600 mg/kg/day, 600 to 700mg/kg/day, 700 to 800 mg/kg/day, 800 to 900 mg/kg/day, 900 to 1000mg/kg/day or a combination thereof. Typical dosages of the any one ormore of the ApoB100 peptides and/or a combinations thereof, or analogs,pharmaceutical equivalents or a peptidomimetics thereof can be in theranges recommended by the manufacturer where known therapeutic compoundsare used, and also as indicated to the skilled artisan by the in vitroresponses or responses in animal models. Such dosages typically can bereduced by up to about an order of magnitude in concentration or amountwithout losing relevant biological activity. The actual dosage candepend upon the judgment of the physician, the condition of the patient,and the effectiveness of the therapeutic method based, for example, onthe in vitro responsiveness of relevant cultured cells or histoculturedtissue sample, such as biopsied malignant tumors, or the responsesobserved in the appropriate animal models. In various embodiments, theany one or more of the ApoB100 peptides and/or a combinations thereof,or analogs, pharmaceutical equivalents or a peptidomimetics thereof maybe administered once a day (SID/QD), twice a day (BID), three times aday (TID), four times a day (QID), or more, so as to administer aneffective amount to the subject, where the effective amount is any oneor more of the doses described herein.

In various embodiments, the therapeutically or prophylacticallyeffective amount of the antibodies or fragments thereof specific toApoB100, for use with the methods described herein is any one or more ofabout 0.01 to 0.05 μg/kg/day, 0.05-0.1 μg/kg/day, 0.1 to 0.5 μg/kg/day,0.5 to 5 μg/kg/day, 0.5 to 1 μg/kg/day, 1 to 5 μg/kg/day, 5 to 10μg/kg/day, 10 to 20 μg/kg/day, 20 to 50 μg/kg/day, 50 to 100 μg/kg/day,100 to 150 μg/kg/day, 150 to 200 μg/kg/day, 200 to 250 μg/kg/day, 250 to300 μg/kg/day, 300 to 350 μg/kg/day, 350 to 400 μg/kg/day, 400 to 500μg/kg/day, 500 to 600 μg/kg/day, 600 to 700 μg/kg/day, 700 to 800μg/kg/day, 800 to 900 μg/kg/day, 900 to 1000 μg/kg/day, 0.01 to 0.05mg/kg/day, 0.05-0.1 mg/kg/day, 0.1 to 0.5 mg/kg/day, 0.5 to 1 mg/kg/day,1 to 5 mg/kg/day, 5 to 10 mg/kg/day, 10 to 15 mg/kg/day, 15 to 20mg/kg/day, 20 to 50 mg/kg/day, 50 to 100 mg/kg/day, 100 to 200mg/kg/day, 200 to 300 mg/kg/day, 300 to 400 mg/kg/day, 400 to 500mg/kg/day, 500 to 600 mg/kg/day, 600 to 700 mg/kg/day, 700 to 800mg/kg/day, 800 to 900 mg/kg/day, 900 to 1000 mg/kg/day or a combinationthereof. Typical dosages of the antibodies can be in the rangesrecommended by the manufacturer where known therapeutic compounds areused, and also as indicated to the skilled artisan by the in vitroresponses or responses in animal models. Such dosages typically can bereduced by up to about an order of magnitude in concentration or amountwithout losing relevant biological activity. The actual dosage candepend upon the judgment of the physician, the condition of the patient,and the effectiveness of the therapeutic method based, for example, onthe in vitro responsiveness of relevant cultured cells or histoculturedtissue sample, such as biopsied malignant tumors, or the responsesobserved in the appropriate animal models. In various embodiments, theantibodies may be administered once a day (SID/QD), twice a day (BID),three times a day (TID), four times a day (QID), or more, so as toadminister an effective amount to the subject, where the effectiveamount is any one or more of the doses described herein.

In various embodiments, the subject is selected from the groupconsisting of human, non-human primate, monkey, ape, dog, cat, cow,horse, rabbit, mouse and rat.

Diagnostic Methods

Further provided herein are assays for diagnosing SLE in a subject inneed thereof. The assays include obtaining a sample from the subject;assaying the sample to determine the level of autoantibodies againstApoB100; and determining that the subject has increased likelihood ofSLE if the level of the autoantibodies is decreased relative to areference value, or determining that the subject has decreasedlikelihood of SLE if the level of autoantibodies is increased relativeto a reference value. In some embodiments, the method may furthercomprise selecting or prescribing a therapy for SLE.

Also provided herein are assays for determining likelihood ofcardiovascular disease in a subject having or suspected of having SLE.The assays include obtaining a sample from the subject; assaying thesample to determine the level of autoantibodies against ApoB100; anddetermining that the subject has increased likelihood of cardiovasculardisease if the level of the autoantibodies is decreased relative to areference value, or determining that the subject has decreasedlikelihood of cardiovascular disease if the level of autoantibodies isincreased relative to a reference value. In some embodiments, the methodmay further comprise selecting or prescribing a therapy forcardiovascular disease.

Also provided herein are assays for determining the efficacy oftreatment for SLE in a subject in need thereof. The assays includeobtaining a sample from the subject undergoing treatment for SLE;assaying the sample to determine the level of autoantibodies againstApoB100; and determining that the treatment is effective if the level ofthe autoantibodies is increased relative to a reference value, ordetermining that the treatment is ineffective if the level ofautoantibodies is decreased or unchanged relative to a reference value.

Also provided herein are assays for determining the efficacy oftreatment for cardiovascular diseases in subject with SLE. The assaysinclude obtaining a sample from the subject undergoing treatment forcardiovascular diseases; assaying the sample to determine the level ofautoantibodies against ApoB100; and determining that the treatment iseffective if the level of the autoantibodies is increased relative to areference value, or determining that the treatment is ineffective if thelevel of autoantibodies is decreased or unchanged relative to areference value.

Further provided herein is an assay for diagnosing SLE in a subject inneed thereof. The assay includes obtaining a sample from the subject;assaying the sample to determine the level of soluble forms of theapoptosis-signaling receptors Fas, TNF-R1, and/or TRAIL-R2; anddetermining that the subject has increased likelihood of having SLE ifthe level of the said apoptosis-signaling receptors is increasedrelative to a reference value, or determining that the subject hasdecreased likelihood of having SLE if the level of saidapoptosis-signaling receptors is decreased relative to a referencevalue.

Further provided herein is an assay for determining the efficacy oftreatment for SLE in a subject in need thereof. The assay includesobtaining a sample from the subject; assaying the sample to determinethe level of soluble forms of the apoptosis-signaling receptors Fas,TNF-R1, and/or TRAIL-R2; and determining that the treatment is effectiveif the level of the said apoptosis-signaling receptors is decreasedrelative to a reference value, or determining that the treatment is lesseffective or not effective if the level of said apoptosis-signalingreceptors is increased relative to a reference value

Also provided herein is an assay for determining likelihood ofcardiovascular disease in a subject having or suspected of having SLE.The assay includes obtaining a sample from the subject; assaying thesample to determine the level of soluble forms of theapoptosis-signaling receptors Fas, TNF-R1, and/or TRAIL-R2; anddetermining that the subject has increased likelihood of cardiovasculardisease if the level of said apoptosis-signaling receptors is increasedrelative to a reference value, or determining that the subject hasdecreased likelihood of cardiovascular disease if the level of saidapoptosis-signaling receptors is decreased relative to a referencevalue.

In various embodiments, assaying comprises using is immunoassays.Exemplary embodiments of immunoassays include but are not limited to anyone or more of ELISA, RIA, Western blotting, Southern blotting, orcombinations thereof.

In some embodiments of the assays described herein, the sample is anyone or more of blood, plasma, urine, tissue or combinations thereof. Insome embodiments of the assays described herein, the sample is obtainedbefore, during or after treatment for SLE.

In one embodiment of the assays described herein, the subject is human.

In one embodiment of the assays described herein, cardiovascular diseaseis atherosclerosis. In an exemplary embodiment, the atherosclerosis isaccelerated atherosclerosis. In some embodiments, the sample is obtainedbefore, during and/or after treatment for cardiovascular diseases insubject having or suspected of having SLE.

Any suitable immunoassay method may be utilized, including those whichare commercially available, to determine the level ApoB100 specificautoantibodies measured according to the invention. Extensive discussionof the known immunoassay techniques is not required here since these areknown to those of skill in the art. Typical suitable immunoassaytechniques include sandwich enzyme-linked immunoassays (ELISA),radioimmunoassays (RIA), competitive binding assays, homogeneous assays,heterogeneous assays, etc. Various known immunoassay methods arereviewed, e.g., in Methods in Enzymology, 70, pp. 30-70 and 166-198(1980).

In the assays of the invention, “sandwich-type” assay formats can beused. Some examples of such sandwich-type assays are described in byU.S. Pat. No. 4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 toTom, et al. An alternative technique is the “competitive-type” assay. Ina competitive assay, the labeled probe is generally conjugated with amolecule that is identical to, or an analog of, the analyte. Thus, thelabeled probe competes with the analyte of interest for the availablereceptive material. Competitive assays are typically used for detectionof analytes such as haptens, each hapten being monovalent and capable ofbinding only one antibody molecule. Examples of competitive immunoassaydevices are described in U.S. Pat. No. 4,235,601 to Deutsch, et al.,U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 toBuechler, et al.

The antibodies can be labeled. In some embodiments, the detectionantibody is labeled by covalently linking to an enzyme, label with afluorescent compound or metal, label with a chemiluminescent compound.For example, the detection antibody can be labeled with catalase and theconversion uses a colorimetric substrate composition comprises potassiumiodide, hydrogen peroxide and sodium thiosulphate; the enzyme can bealcohol dehydrogenase and the conversion uses a colorimetric substratecomposition comprises an alcohol, a pH indicator and a pH buffer,wherein the pH indicator is neutral red and the pH buffer isglycine-sodium hydroxide; the enzyme can also be hypoxanthine oxidaseand the conversion uses a colorimetric substrate composition comprisesxanthine, a tetrazolium salt and 4,5-dihydroxy-1,3-benzene disulphonicacid. In one embodiment, the detection antibody is labeled by covalentlylinking to an enzyme, label with a fluorescent compound or metal, orlabel with a chemiluminescent compound.

Direct and indirect labels can be used in immunoassays. A direct labelcan be defined as an entity, which in its natural state, is visibleeither to the naked eye or with the aid of an optical filter and/orapplied stimulation, e.g., ultraviolet light, to promote fluorescence.Examples of colored labels which can be used include metallic solparticles, gold sol particles, dye sol particles, dyed latex particlesor dyes encapsulated in liposomes. Other direct labels includeradionuclides and fluorescent or luminescent moieties. Indirect labelssuch as enzymes can also be used according to the invention. Variousenzymes are known for use as labels such as, for example, alkalinephosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphatedehydrogenase, lactate dehydrogenase and urease. For a detaileddiscussion of enzymes in immunoassays see Engvall, Enzyme ImmunoassayELISA and EMIT, Methods of Enzymology, 70, 419-439 (1980).

The antibody can be attached to a surface. Examples of useful surfaceson which the antibody can be attached for the purposes of detecting thedesired antigen include nitrocellulose, PVDF, polystyrene, and nylon.The surface or support may also be a porous support (see, e.g., U.S.Pat. No. 7,939,342). The assays can be carried out in various assaydevice formats including those described in U.S. Pat. Nos. 4,906,439;5,051,237 and 5,147,609 to PB Diagnostic Systems, Inc.

Reference Values

In various embodiments of the assays described herein, the referencevalue is based on the levels of ApoB100 specific autoantibodies. In oneembodiment, the reference value is the mean or median level ofautoantibodies against ApoB100 in a population of subjects that do nothave SLE. In another embodiment, the reference value is the mean ormedian level of autoantibodies against ApoB100 in a sample obtained fromthe subject at a different time point (for example, prior to startingtreatment). In a further embodiment, the reference value is the mean ormedian level of autoantibodies against ApoB100 in a population ofsubjects that have SLE and have undergone or are undergoing treatmentfor SLE. In an additional embodiment, the reference value is the mean ormedian level of autoantibodies against ApoB100 in a population ofsubjects that have SLE and have undergone or are undergoing treatmentfor SLE and have not undergone or are not undergoing treatment forcardiovascular diseases. In an additional embodiment, the referencevalue is the mean or median level of autoantibodies against ApoB100 in apopulation of subjects that have SLE and have undergone or areundergoing treatment for SLE and have undergone or are undergoingtreatment for cardiovascular diseases.

In various embodiments, the levels of ApoB100 specific autoantibodies inthe subject compared to the reference values is decreased by at least orabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In variousembodiments, the levels of ApoB100 specific autoantibodies in thesubject compared to the reference values is decreased by at least orabout 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold,25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold,65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold or100-fold

Peptides of ApoB100

Specific immunogenic epitopes of ApoB100 were characterized and apeptide library including 302 peptides, each about 20 amino acidresidues in length, covering the complete 4563 amino acid sequence ofhuman ApoB100 was produced. The peptides were produced with a 5 aminoacid overlap to cover all sequences at break points. Peptides werenumbered 1-302 starting at the N-terminal of ApoB100 as indicated inTable 1 below.

TABLE 1 Peptide Sequence Apolipoprotein B SEQ ID NO P1:EEEML ENVSL VCPKD ATRFK aa 1-20 SEQ ID NO: 1 P2: ATRFK HLRKY TYNYE AESSSaa 16-35 SEQ ID NO: 2 P3: AESSS GVPGT ADSRS ATRIN aa 31-50 SEQ ID NO: 3P4: ATRIN CKVEL EVPQL CSFIL aa 46-65 SEQ ID NO: 4 P5:CSFIL KTSQC TLKEV YGFNP aa 61-80 SEQ ID NO: 5 P6:YGFNP EGKAL LKKTK NSEEF aa 76-95 SEQ ID NO: 6 P7:NSEEF AAAMS RYELK LAIPE aa 91-110 SEQ ID NO: 7 P8:LAIPE GKQVF LYPEK DEPTY aa 106-125 SEQ ID NO: 8 P9:DEPTY ILNIK RGIIS ALLVP aa 121-140 SEQ ID NO: 9 P10:ALLVP PETEE AKQVL FLDTV aa 136-155 SEQ ID NO: 10 P11:FLDTV YGNCS THFTV KTRKG aa 151-170 SEQ ID NO: 11 P12:KTRKG NVATE ISTER DLGQC aa 166-185 SEQ ID NO: 12 P13:DLGQC DRFKP IRTGI SPLAL aa 181-200 SEQ ID NO: 13 P14:SPLAL IKGMT RPLST LISSS aa 196-215 SEQ ID NO: 14 P15:LISSS QSCQY TLDAK RKHVA aa 211-230 SEQ ID NO: 15 P16:RKHVA EAICK EQHLF LPFSY aa 226-245 SEQ ID NO: 16 P17:LPFSY NNKYG MVAQV TQTLK aa 241-260 SEQ ID NO: 17 P18:TQTLK LEDTP KINSR FFGEG aa 256-275 SEQ ID NO: 18 P19:FFGEG TKKMG LAFES TKSTS aa 271-290 SEQ ID NO: 19 P20:TKSTS PPKQA EAVLK TLQEL aa 286-305 SEQ ID NO: 20 P21:TLQEL KKLTI SEQNI QRANL aa 301-320 SEQ ID NO: 21 P22:QRANL FNKLV TELRG LSDEA aa 316-335 SEQ ID NO: 22 P23:LSDEA VTSLL PQLIE VSSPI aa 331-350 SEQ ID NO: 23 P24:VSSPI TLQAL VQCGQ PQCST aa 346-365 SEQ ID NO: 24 P25:PQCST HILQW LKRVH ANPLL aa 361-380 SEQ ID NO: 25 P26:ANPLL IDVVT YLVAL IPEPS aa 376-395 SEQ ID NO: 26 P27:IPEPS AQQLR EIFNM ARDQR aa 391-410 SEQ ID NO: 27 P28:ARDQR SRATL YALS AVNNY aa 406-425 SEQ ID NO: 28 P29:AVNNY HKTNP TGTQE LLDIA aa 421-440 SEQ ID NO: 29 P30:LLDIA NYLME QIQDD CTGDE aa 436-455 SEQ ID NO: 30 P31:CTGDE DYTYL ILRVI GNMGQ aa 451-470 SEQ ID NO: 31 P32:GNMGQ TMEQL TPELK SSILK aa 466-485 SEQ ID NO: 32 P33:SSILK CVQST KPSLM IQKAA aa 481-500 SEQ ID NO: 33 P34:IQKAA IQALR KMEPK DKDQE aa 496-515 SEQ ID NO: 34 P35:DKDQE VLLQT FLDDA SPGDK aa 511-530 SEQ ID NO: 35 P36:SPGDK RLAAY LMLMRSPSQA aa 526-545 SEQ ID NO: 36 P37:SPSQA DINKIVQILP WEQNE aa 541-560 SEQ ID NO: 37 P38:WEQNE QVKNF VASHI ANILN aa 556-575 SEQ ID NO: 38 P39:ANILN SEELD IQDLK KLVKE aa 571-590 SEQ ID NO: 39 P40:KLVKE ALKES QLPTV MDFRK aa 586-605 SEQ ID NO: 40 P41:MDFRK FSRNY QLYKS VSLPS aa 601-620 SEQ ID NO: 41 P42:VSLPS LDPAS AKIEG NLIFD aa 616-635 SEQ ID NO: 42 P43:NLIFD PNNYL PKESM LKTTL aa 631-650 SEQ ID NO: 43 P44:LKTTL TAFGF ASADL IEIGL aa 646-665 SEQ ID NO: 44 P45:IEIGL EGKGF EPTLE ALFGK aa 661-680 SEQ ID NO: 45 P46:ALFGK QGFFP DSVNK ALYWV aa 676-695 SEQ ID NO: 46 P47:ALYWV NGQVP DGVSK VLVDH aa 691-710 SEQ ID NO: 47 P48:VLVDH FGYTK DDKHE QDMVN aa 706-725 SEQ ID NO: 48 P49:QDMVN GIMLS VEKLI KDLKS aa 721-740 SEQ ID NO: 49 P50:KDLKS KEVPE ARAYL RILGE aa 736-755 SEQ ID NO: 50 P51:RILGE ELGFA SLHDL QLLGK aa 751-770 SEQ ID NO: 51 P52:QLLGK LLLMG ARTLQ GIPQM aa 766-785 SEQ ID NO: 52 P53:GIPQM IGEVI RKGSK NDFFL aa 781-800 SEQ ID NO: 53 P54:NDFFL HYIFM ENAFE LPTGA aa 796-815 SEQ ID NO: 54 P55:LPTGA GLQLQ ISSSG VIAPG aa 811-830 SEQ ID NO: 55 P56:VIAPG AKAGV KLEVA NMQAE aa 826-845 SEQ ID NO: 56 P57:NMQAE LVAKP SVSVE FVTNM aa 841-860 SEQ ID NO: 57 P58:FVTNM GIIIP DFARS GVQMN aa 856-875 SEQ ID NO: 58 P59:GVQMN TNFFH ESGLE AHVAL aa 871-890 SEQ ID NO: 59 P60:AHVAL KAGKL KFIIP SPKRP aa 886-905 SEQ ID NO: 60 P61:SPKRP VKLLS GGNTL HLVST aa 901-920 SEQ ID NO: 61 P62:HLVST TKTEV IPPLI ENRQS aa 916-935 SEQ ID NO: 62 P63:ENRQS WSVCKQVFPG LNYCT aa 931-950 SEQ ID NO: 63 P64:LNYCT SGAYS NASST DSASY aa 946-965 SEQ ID NO: 64 P65:DSASY YPLTG DTRLE LELRP aa 961-980 SEQ ID NO: 65 P66:LELRP TGEIE QYSVS ATYEL aa 976-995 SEQ ID NO: 66 P67:ATYEL QREDR ALVDT LKFVT aa 991-1010 SEQ ID NO: 67 P68:LKFVT QAEGA KQTEA TMTFK aa 1006-1025 SEQ ID NO: 68 P69:TMTFK YNRQS MTLSS EVQIP aa 1021-1040 SEQ ID NO: 69 P70:EVQIP DFDVD LGTIL RVNDE aa 1036-1055 SEQ ID NO: 70 P71:RVNDE STEGK TSYRL TLDIQ aa 1051-1070 SEQ ID NO: 71 P72:TLDIQ NKKIT EVALM GHLSC aa 1066-1085 SEQ ID NO: 72 P73:GHLSC DTKEE RKIKG VISIP aa 1081-1100 SEQ ID NO: 73 P74:VISIP RLQAE ARSEI LAHWS aa 1096-1115 SEQ ID NO: 74 P75:LAHWS PAKLL LQMDS SATAY aa 1111-1130 SEQ ID NO: 75 P76:SATAY GSTVS KRVAW HYDEE aa 1126-1145 SEQ ID NO: 76 P77:HYDEE KIEFE WNTGT NVDTK aa 1141-1160 SEQ ID NO: 77 P78:NVDTK KMTSN FPVDL SDYPK aa 1156-1175 SEQ ID NO: 78 P79:SDYPK SLHMY ANRLL DHRVP aa 1171-1190 SEQ ID NO: 79 P80:DHRVP ETDMT FRHVG SKLIV aa 1186-1205 SEQ ID NO: 80 P81:SKLIV AMSSW LQKAS GSLPY aa 1201-1220 SEQ ID NO: 81 P82:GSLPY TQTLQ DHLNS LKEFN aa 1216-1235 SEQ ID NO: 82 P83:LKEFN LQNMG LPDFH IPENL aa 1231-1250 SEQ ID NO: 83 P84:IPENL FLKSD GRVKY TLNKN aa 1246-1260 SEQ ID NO: 84 P85:TLNKN SLKIE IPLPF GGKSS aa 1261-1280 SEQ ID NO: 85 P86:GGKSS RDLKM LETVR TPALH aa 1276-1295 SEQ ID NO: 86 P87:TPALH FKSVG FHLPS REFQV aa 1291-1310 SEQ ID NO: 87 P88:REFQV PTFTI PKLYQ LQVPL aa 1306-1325 SEQ ID NO: 88 P89:LQVPL LGVLD LSTNV YSNLY aa 1321-1340 SEQ ID NO: 89 P90:YSNLY NWSAS YSGGN TSTDH aa 1336-1355 SEQ ID NO: 90 P91:TSTDH FSLRA RYHMK ADSVV aa 1351-1370 SEQ ID NO: 91 P92:ADSVV DLLSY NVQGS GETTY aa 1366-1385 SEQ ID NO: 92 P93:GETTY DHKNT FTLSC DGSLR aa 1381-1400 SEQ ID NO: 93 P94:DGSLR HKFLD SNIKF SHVEK aa 1396-1415 SEQ ID NO: 94 P95:SHVEK LGNNP VSKGL LIFDA aa 1411-1430 SEQ ID NO: 95 P96:LIFDA SSSWG PQMSA SVHLD aa 1426-1445 SEQ ID NO: 96 P97:SVHLD SKKKQ HLFVK EVKID aa 1441-1460 SEQ ID NO: 97 P98:EVKID GQFRV SSFYA KGTYG aa 1456-1475 SEQ ID NO: 98 P99:KGTYG LSCQR DPNTG RLNGE aa 1471-1490 SEQ ID NO: 99 P100:RLNGE SNLRF NSSYL QGTNQ aa 1486-1505 SEQ ID NO: 100 P101:QGTNQ ITGRY EDGTL SLTST aa 1501-1520 SEQ ID NO: 101 P102:SLTST SDLQS GIIKN TASLK aa 1516-1535 SEQ ID NO: 102 P103:TASLK YENYE LTLKS DTNGK aa 1531-1550 SEQ ID NO: 103 P104:DTNGK YKNFA TSNKM DMTFS aa 1546-1565 SEQ ID NO: 104 P105:DMTFS KQNAL LRSEY QADYE aa 1561-1580 SEQ ID NO: 105 P106:QADYE SLRFF SLLSG SLNSH aa 1576-1595 SEQ ID NO: 106 P107:SLNSH GLELN ADILG TDKIN aa 1591-1610 SEQ ID NO: 107 P108:TDKIN SGAHK ATLRI GQDGI aa 1606-1625 SEQ ID NO: 108 P109:GQDGI STSAT TNLKCSLLVL aa 1621-1640 SEQ ID NO: 109 P110:SLLVL ENELN AELGL SGASM aa 1636-1655 SEQ ID NO: 110 P111:SGASM KLTTN GRFRE HNAKF aa 1651-1670 SEQ ID NO: 111 P112:HNAKF SLDGK AALTE LSLGS aa 1666-1685 SEQ ID NO: 112 P113:LSLGS AYQAM ILGVD SKNIF aa 1681-1700 SEQ ID NO: 113 P114:SKNIF NFKVS QEGLK LSNDM aa 1696-1715 SEQ ID NO: 114 P115:LSNDM MGSYA EMKFD HTNSL aa 1711-1730 SEQ ID NO: 115 P116:HTNSL NIAGL SLDFS SKLDN aa 1726-1745 SEQ ID NO: 116 P117:SKLDN IYSSD KFYKQ TVNLQ aa 1741-1760 SEQ ID NO: 117 P118:TVNLQ LQPYS LVTTL NSDLK aa 1756-1775 SEQ ID NO: 118 P119:NSDLK YNALD LTNNG KLRLE aa 1771-1790 SEQ ID NO: 119 P120:KLRLE PLKLH VAGNL KGAYQ aa 1786-1805 SEQ ID NO: 120 P121:KGAYQ NNEIK HIYAI SSAAL aa 1801-1820 SEQ ID NO: 121 P122:SSAAL SASYK ADTVA KVQGV aa 1816-1835 SEQ ID NO: 122 P123:KVQGV EFSHR LNTDI AGLAS aa 1831-1850 SEQ ID NO: 123 P124:AGLAS AIDMS TNYNS DSLHF aa 1846-1865 SEQ ID NO: 124 P125:DSLHF SNVFR SVMAP FTMTI aa 1861-1880 SEQ ID NO: 125 P126:FTMTI DAHTN GNGKL ALWGE aa 1876-1895 SEQ ID NO: 126 P127:ALWGE HTGQL YSKFL LKAEP aa 1891-1910 SEQ ID NO: 127 P128:LKAEP LAFTF SHDYK GSTSH aa 1906-1925 SEQ ID NO: 128 P129:GSTSH HLVSR KSISA ALEHK aa 1921-1940 SEQ ID NO: 129 P130:ALEHK VSALL TPAEQ TGTWK aa 1936-1955 SEQ ID NO: 130 P131:TGTWK LKTQF NNNEY SQDLD aa 1951-1970 SEQ ID NO: 131 P132:SQDLD AYNTK DKIGV ELTGR aa 1966-1985 SEQ ID NO: 132 P133:ELTGR TLADL TLLDS PIKVP aa 1981-2000 SEQ ID NO: 133 P134:PIKVP LLLSE PINII DALEM aa 1996-2015 SEQ ID NO: 134 P135:DALEM RDAVE KPQEF TIVAF aa 2011-2030 SEQ ID NO: 135 P136:TIVAF VKYDK NQDVH SINLP aa 2026-2045 SEQ ID NO: 136 P137:SINLP FFETL QEYFE RNRQT aa 2041-2060 SEQ ID NO: 137 P138:RNRQT IIVVV ENVQR NLKHI aa 2056-2075 SEQ ID NO: 138 P139:NLKHI NIDQF VRKYR AALGK aa 2071-2090 SEQ ID NO: 139 P140:AALGK LPQQA NDYLN SFNWE aa 2086-2105 SEQ ID NO: 140 P141:SFNWE RQVSH AKEKL TALTK aa 2101-2120 SEQ ID NO: 141 P142:TALTK KYRIT ENDIQ IALDD aa 2116-2135 SEQ ID NO: 142 P143:IALDD AKINF NEKLS QLQTY aa 2131-2150 SEQ ID NO: 143 P144:QLQTY MIQFD QYIKD SYDLH aa 2146-2165 SEQ ID NO: 144 P145:SYDLH DLKIA IANII DEIIE aa 2161-2180 SEQ ID NO: 145 P146:DEIIE KLKSL DEHYH IRVNL aa 2176-2195 SEQ ID NO: 146 P147:IRVNL VKTIH DLHLF IENID aa 2191-2210 SEQ ID NO: 147 P148:IENID FNKSG SSTAS WIQNV aa 2206-2225 SEQ ID NO: 148 P149:WIQNV DTKYQ IRIQI QEKLQ aa 2221-2240 SEQ ID NO: 149 P150:QEKLQ QLKRH IQNID IQHLA aa 2236-2255 SEQ ID NO: 150 P151:IQHLA GKLKQ HIEAI DVRVL aa 2251-2270 SEQ ID NO: 151 P152:DVRVL LDQLG TTISF ERIND aa 2266-2285 SEQ ID NO: 152 P153:ERIND VLEHV KHFVI NLIGD aa 2281-2300 SEQ ID NO: 153 P154:NLIGD FEVAE KINAF RAKVH aa 2296-2315 SEQ ID NO: 154 P155:RAKVH ELIER YEVDQ QIQVL aa 2311-2330 SEQ ID NO: 155 P156:QIQVL MDKLV ELTHQ YKLKE aa 2326-2345 SEQ ID NO: 156 P157:YKLKE TIQKL SNVLQ QVKIK aa 2341-2360 SEQ ID NO: 157 P158:QVKIK DYFEK LVGFI DDAVK aa 2356-2375 SEQ ID NO: 158 P159:DDAVK KLNEL SFKTF IEDVN aa 2371-2390 SEQ ID NO: 159 P160:IEDVN KFLDM LIKKL KSFDY aa 2386-2405 SEQ ID NO: 160 P161:KSFDY HQFVD ETNDK IREVT aa 2401-2420 SEQ ID NO: 161 P162:IREVT QRLNG EIQAL ELPQK aa 2416-2435 SEQ ID NO: 162 P163:ELPQK AEALK LFLEE TKATV aa 2431-2450 SEQ ID NO: 163 P164:TKATV AVYLE SLQDT KITLI aa 2446-2465 SEQ ID NO: 164 P165:KITLI INWLQ EALSS ASLAH aa 2461-2480 SEQ ID NO: 165 P166:ASLAH MKAKF RETLE DTRDR aa 2476-2495 SEQ ID NO: 166 P167:DTRDR MYQMD IQQEL QRYLS aa 2491-2510 SEQ ID NO: 167 P168:QRYLS LVGQV YSTLV TYISD aa 2506-2515 SEQ ID NO: 168 P169:TYISD WWTLA AKNLT DFAEQ aa 2521-2540 SEQ ID NO: 169 P170:DFAEQ YSIQD WAKRM KALVE aa 2536-2555 SEQ ID NO: 170 P171:KALVE QGFTV PEIKT ILGTM aa 2551-2570 SEQ ID NO: 171 P172:ILGTM PAFEV SLQAL QKATF aa 2566-2585 SEQ ID NO: 172 P173:QKATF QTPDF IVPLT DLRIP aa 2581-2600 SEQ ID NO: 173 P174:DLRIP SVQIN FKDLK NIKIP aa 2596-2615 SEQ ID NO: 174 P175:NIKIP SRFST PEFTI LNTFH aa 2611-2630 SEQ ID NO: 175 P176:LNTFH IPSFT IDFVE MKVKI aa 2626-2645 SEQ ID NO: 176 P177:MKVKI IRTID QMQNS ELQWP aa 2641-2660 SEQ ID NO: 177 P178:ELQWP VPDIY LRDLK VEDIP aa 2656-2675 SEQ ID NO: 178 P179:VEDIP LARIT LPDFR LPEIA aa 2671-2690 SEQ ID NO: 179 P180:LPEIA IPEFI IPTLN LNDFQ aa 2686-2705 SEQ ID NO: 180 P181:LNDFQ VPDLH IPEFQ LPHIS aa 2701-2720 SEQ ID NO: 181 P182:LPHIS HTIEV PTFGK LYSIL aa 2716-2735 SEQ ID NO: 182 P183:LYSIL KIQSP LFTLD ANADI aa 2731-2750 SEQ ID NO: 183 P184:ANADI GNGTT SANEA GIAAS aa 2746-2765 SEQ ID NO: 184 P185:GIAAS ITAKG ESKLE VLNFD aa 2761-2780 SEQ ID NO: 185 P186:VLNFD FQANA QLSNP KINPL aa 2776-2795 SEQ ID NO: 186 P187:KINPL ALKES VKFSS KYLRT aa 2791-2810 SEQ ID NO: 187 P188:KYLRT EHGSE MLFFG NAIEG aa 2806-2825 SEQ ID NO: 188 P189:NAIEG KSNTV ASLHT EKNTL aa 2821-2840 SEQ ID NO: 189 P190:EKNTL ELSNG VIVKI NNQLT aa 2836-2855 SEQ ID NO: 190 P191:NNQLT LDSNT KYFHK LNIPK aa 2851-2870 SEQ ID NO: 191 P192:LNIPK LDFSS QADLR NEIKT aa 2866-2885 SEQ ID NO: 192 P193:NEIKT LLKAG HIAWT SSGKG aa 2881-2900 SEQ ID NO: 193 P194:SSGKG SWKWA CPRFS DEGTH aa 2896-2915 SEQ ID NO: 194 P195:DEGTH ESQIS FTIEG PLTSF aa 2911-2930 SEQ ID NO: 195 P196:PLTSF GLSNK INSKH LRVNQ aa 2926-2945 SEQ ID NO: 196 P197:LRVNQ NLVYE SGSLN FSKLE aa 2941-2960 SEQ ID NO: 197 P198:FSKLE IQSQV DSQHV GHSVL aa 2956-2975 SEQ ID NO: 198 P199:GHSVL TAKGM ALFGE GKAEF aa 2971-2990 SEQ ID NO: 199 P200:GKAEF TGRHD AHLNG KVIGT aa 2986-3005 SEQ ID NO: 200 P201:KVIGT LKNSL FFSAQ PFEIT aa 3001-3020 SEQ ID NO: 201 P202:PFEIT ASTNN EGNLK VRFPL aa 3016-3035 SEQ ID NO: 202 P203:VRFPL RLTGK IDFLN NYALF aa 3031-3050 SEQ ID NO: 203 P204:NYALF LSPSA QQASW QVSAR aa 3046-3065 SEQ ID NO: 204 P205:QVSAR FNQYK YNQNF SAGNN aa 3061-3080 SEQ ID NO: 205 P206:SAGNN ENIME AHVGI NGEAN aa 3076-3095 SEQ ID NO: 206 P207:NGEAN LDFLN IPLTI PEMRL aa 3091-3110 SEQ ID NO: 207 P208:PEMRL PYTII TTPPL KDFSL aa 3106-3125 SEQ ID NO: 208 P209:KDFSL WEKTG LKEFL KTTKQ aa 3121-3140 SEQ ID NO: 209 P210:KTTKQ SFDLS VKAQY KKNKH aa 3136-3155 SEQ ID NO: 210 P211:KKNKH RHSIT NPLAV LCEFI aa 3151-3170 SEQ ID NO: 211 P212:LCEFI SQSIK SFDRH FEKNR aa 3166-3185 SEQ ID NO: 212 P213:FEKNR NNALD FVTKS YNETK aa 3181-3200 SEQ ID NO: 213 P214:YNETK IKFDK YKAEK SHDEL aa 3196-3215 SEQ ID NO: 214 P215:SHDEL PRTFQ IPGYT VPVVN aa 3211-3230 SEQ ID NO: 215 P216:VPVVN VEVSP FTIEM SAFGY aa 3226-3245 SEQ ID NO: 216 P217:SAFGY VFPKA VSMPS FSILG aa 3241-3260 SEQ ID NO: 217 P218:FSILG SDVRV PSYTL ILPSL aa 3256-3275 SEQ ID NO: 218 P219:ILPSL ELPVL HVPRN LKLSL aa 3271-3290 SEQ ID NO: 219 P220:LKLSL PHFKE LCTIS HIFIP aa 3286-3305 SEQ ID NO: 220 P221:HIFIP AMGNI TYDFS FKSSV aa 3301-3320 SEQ ID NO: 221 P222:FKSSV ITLNT NAELF NQSDI aa 3316-3335 SEQ ID NO: 222 P223:NQSDI VAHLL SSSSS VIDAL aa 3331-3350 SEQ ID NO: 223 P224:VIDAL QYKLE GTTRL TRKRG aa 3346-3365 SEQ ID NO: 224 P225:TRKRG LKLAT ALSLS NKFVE aa 3361-3380 SEQ ID NO: 225 P226:NKFVE GSHNS TVSLT TKNME aa 3376-3395 SEQ ID NO: 226 P227:TKNME VSVAK TTKAE IPILR aa 3391-3410 SEQ ID NO: 227 P228:IPILR MNFKQ ELNGN TKSKP aa 3406-3425 SEQ ID NO: 228 P229:TKSKP TVSSS MEFKY DFNSS aa 3421-3440 SEQ ID NO: 229 P230:DFNSS MLYST AKGAV DHKLS aa 3436-3455 SEQ ID NO: 230 P231:DHKLS LESLT SYFSI ESSTK aa 3451-3470 SEQ ID NO: 231 P232:ESSTK GDVKG SVLSR EYSGT aa 3466-3485 SEQ ID NO: 232 P233:EYSGT IASEA NTYLN SKSTR aa 3481-3500 SEQ ID NO: 233 P234:SKSTR SSVKL QGTSK IDDIW aa 3496-3515 SEQ ID NO: 234 P235:IDDIW NLEVK ENFAG EATLQ aa 3511-3530 SEQ ID NO: 235 P236:EATLQ RIYSL WEHST KNHLQ aa 3526-3545 SEQ ID NO: 236 P237:KNHLQ LEGLF FTNGE HTSKA aa 3541-3560 SEQ ID NO: 237 P238:HTSKA TLELS PWQMS ALVQV aa 3556-3575 SEQ ID NO: 238 P239:ALVQV HASQP SSFHD FPDLG aa 3571-3590 SEQ ID NO: 239 P240:FPDLG QEVAL NANTK NQKIR aa 3586-3605 SEQ ID NO: 240 P241:NQKIR WKNEV RIHSG SFQSQ aa 3601-3620 SEQ ID NO: 241 P242:SFQSQ VELSN DQEKA HLDIA aa 3616-3635 SEQ ID NO: 242 P243:HLDIA GSLEG HLRFL KNIIL aa 3631-3650 SEQ ID NO: 243 P244:KNIIL PVYDK SLWDF LKLDV aa 3646-3665 SEQ ID NO: 244 P245:LKLDV TTSIG RRQHL RVSTA aa 3661-3680 SEQ ID NO: 245 P246:RVSTA FVYTK NPNGY SFSIP aa 3676-3695 SEQ ID NO: 246 P247:SFSIP VKVLA DKFIT PGLKL aa 3691-3710 SEQ ID NO: 247 P248:PGLKL NDLNS VLVMP TFHVP aa 3706-3725 SEQ ID NO: 248 P249:TFHVP FTDLQ VPSCK LDFRE aa 3721-3740 SEQ ID NO: 249 P250:LDFRE IQIYK KLRTS SFALN aa 3736-3755 SEQ ID NO: 250 P251:SFALN LPTLP EVKFP EVDVL aa 3751-3770 SEQ ID NO: 251 P252:EVDVL TKYSQ PEDSL IPFFE aa 3766-3785 SEQ ID NO: 252 P253:IPFFEITVPE SQLTV SQFTL aa 3781-3800 SEQ ID NO: 253 P254:SQFTL PKSVS DGIAA LDLNA aa 3796-3815 SEQ ID NO: 254 P255:LDLNA VANKI ADFEL PTIIV aa 3811-3830 SEQ ID NO: 255 P256:PTIIV PEQTI EIPSI KFSVP aa 3826-3845 SEQ ID NO: 256 P257:KFSVP AGIVI PSFQA LTARF aa 3841-3860 SEQ ID NO: 257 P258:LTARF EVDSP VYNAT WSASL aa 3856-3875 SEQ ID NO: 258 P259:WSASL KNKAD YVETV LDSTC aa 3871-3890 SEQ ID NO: 259 P260:LDSTC SSTVQ FLEYE LNVLG aa 3886-3905 SEQ ID NO: 260 P261:LNVLG THKIE DGTLA SKTKG aa 3901-3920 SEQ ID NO: 261 P262:SKTKG TLAHR DFSAE YEEDG aa 3916-3935 SEQ ID NO: 262 P263:YEEDG KFEGL QEWEG KAHLN aa 3931-3950 SEQ ID NO: 263 P264:KAHLN IKSPA FTDLH LRYQK aa 3946-3965 SEQ ID NO: 264 P265:LRYQK DKKGI STSAA SPAVG aa 3961-3980 SEQ ID NO: 265 P266:SPAVG TVGMD MDEDD DFSKW aa 3976-3995 SEQ ID NO: 266 P267:DFSKW NFYYS PQSSP DKKLT aa 3991-4010 SEQ ID NO: 267 P268:DKKLT IFKTE LRVRE SDEET aa 4006-4025 SEQ ID NO: 268 P269:SDEET QIKVN WEEEA ASGLL aa 4021-4040 SEQ ID NO: 269 P270:ASGLL TSLKD NVPKA TGVLY aa 4036-4055 SEQ ID NO: 270 P271:TGVLY DYVNK YHWEH TGLTL aa 4051-4070 SEQ ID NO: 271 P272:TGLTL REVSS KLRRN LQNNA aa 4066-4085 SEQ ID NO: 272 P273:LQNNA EWVYQ GAIRQ IDDID aa 4081-4100 SEQ ID NO: 273 P274:IDDID VRFQK AASGT TGTYQ aa 4096-4115 SEQ ID NO: 274 P275:TGTYQ EWKDK AQNLY QELLT aa 4111-4130 SEQ ID NO: 275 P276:QELLT QEGQA SFQGL KDNVF aa 4126-4145 SEQ ID NO: 276 P277:KDNVF DGLVR VTQKF HMKVK aa 4141-4160 SEQ ID NO: 277 P278:HMKVK HLIDS LIDFL NFPRF aa 4156-4175 SEQ ID NO: 278 P279:NFPRF QFPGK PGIYT REELC aa 4171-4190 SEQ ID NO: 279 P280:REELC TMFIR EVGTV LSQVY aa 4186-4205 SEQ ID NO: 280 P281:LSQVY SKVHN GSEIL FSYFQ aa 4201-4220 SEQ ID NO: 281 P282:FSYFQ DLVIT LPFEL RKHKL aa 4216-4235 SEQ ID NO: 282 P283:RKHKL IDVIS MYREL LKDLS aa 4231-4250 SEQ ID NO: 283 P284:LKDLS KEAQE VFKAI QSLKT aa 4246-4265 SEQ ID NO: 284 P285:QSLKT TEVLR NLQDL LQFIF aa 4261-4280 SEQ ID NO: 285 P286:LQFIF QLIED NIKQL KEMKF aa 4276-4295 SEQ ID NO: 286 P287:KEMKF TYLIN YIQDE INTIF aa 4291-4310 SEQ ID NO: 287 P288:INTIF NDYIP YVFKL LKENL aa 4306-4325 SEQ ID NO: 288 P289:LKENL CLNLH KFNEF IQNEL aa 4321-4340 SEQ ID NO: 289 P290:IQNEL QEASQ ELQQI HQYIM aa 4336-4355 SEQ ID NO: 290 P291:HQYIM ALREE YFDPS IVGWT aa 4351-4370 SEQ ID NO: 291 P292:IVGWT VKYYE LEEKI VSLIK aa 4366-4385 SEQ ID NO: 292 P293:VSLIK NLLVA LKDFH SEYIV aa 4381-4400 SEQ ID NO: 293 P294:SEYIV SASNF TSQLS SQVEQ aa 4396-4415 SEQ ID NO: 294 P295:SQVEQ FLHRN IQEYL SILTD aa 4411-4430 SEQ ID NO: 295 P296:SILTD PDGKG KEKIA ELSAT aa 4426-4445 SEQ ID NO: 296 P297:ELSAT AQEII KSQAI ATKKI aa 4441-4460 SEQ ID NO: 297 P298:TKKII SDYHQ QFRYK LQDFS aa 4457-4476 SEQ ID NO: 298 P299:LQDFS DQLSD YYEKF IAESK aa 4472-4491 SEQ ID NO: 299 P300:IAESK RLIDL SIQNY HTFLI aa 4487-4506 SEQ ID NO: 300 P301:HTFLI YITEL LKKLQ STTVM aa 4502-4521 SEQ ID NO: 301 P302:STTVM NPYMK LAPGE LTIIL aa 4517-4536 SEQ ID NO: 302

These peptide sequences were prepared and evaluated for immunogenicactivity as previously described in Patent Publication WO2002080954, thecontents of which is herein incorporated by reference in its entirety.

Antibodies against these peptide sequences were prepared and evaluatedfor immunogenic activity as previously described in WO 2007/025781 andWO 2009/083225, the contents of which are herein incorporated byreference in their entirety.

Specifically, FIG. 2 of WO 2009/083225 describes the amino acid sequenceof the 2D03 heavy chain and the 2D03 light chain, underlining thecomplementarity determining regions (CDRs), which is shown in FIG. 21Band FIG. 21C of the present application. FIG. 21B and FIG. 21C of thepresent application are also seen in FIG. 17 of the presentapplication's priority application, U.S. provisional application No.62/343,601, filed May 31, 2016. Antibody 2D03 refers to orticumab.

Specifically, WO 2007/025781 describes in various locations such aspages 29, 30 and 16 that its invention also includes an antibody thatselectively binds to the oxidised-LDL epitope that is selectively boundby antibody 2D03; a final aspect of the invention provides an antibodycomprising at least one complementarity determining region (CDR) thathas the amino acid sequence of the corresponding CDR of antibody 2D03;that more preferably, the antibody has two or three or four or five CDRsthat have the sequence of the corresponding CDRs of antibody 2D03; thatif the antibody has three or four CDRs that have the sequence of thecorresponding CDRs of antibody 2D03, it is preferred if the antibody hasall three heavy chain or all three light chain CDRs that have thesequence of the corresponding CDRs of antibody 2D03; that thus thisaspect of the invention includes an antibody comprising three lightchain CDRs that have the sequence of the corresponding three light chainCDRs of antibody 2D03, or three heavy chain CDRs that have the sequenceof the corresponding three heavy chain CDRs of antibody 2D03; that yetmore preferably, the antibody comprises three light chain CDRs and threeheavy chain CDRs that have the sequence of the corresponding CDRs ofantibody 2D03; that if the antibody does not comprise all six CDRs thathave the sequence of the corresponding CDRs of antibody 2D03, it ispreferred if some or all of the 1, 2, 3, 4 or 5 “non-identical” CDRscomprise a variant of the sequence of the corresponding CDRs of antibody2D03, (by “a variant” WO 2007/025781 includes the meaning that thevariant has at least 50% sequence identity with the sequence of thecorresponding CDR, more preferably at least 70%, yet more preferably atleast 80% or at least 90% or at least 95%; most preferably, the varianthas 96% or 97% or 98% or 99% sequence identity with the sequence of thecorresponding CDR of antibody 2D03; typically the “variant” CDR sequencehas 5 or 4 or 3 or 2 or only 1 amino acid residue difference from thesequence of the corresponding CDR of antibody 2D03); and that thisaspect of the invention includes antibody 2D03. The incorporation of WO2007/025781 and WO 2009/083225 is also seen in paragraph [0081] (page29) of the present application's priority application, U.S. provisionalapplication No. 62/343,601, filed May 31, 2016. This description isfurther shown in paragraphs below, titled Further Methods and Systems inthe present application.

Preparation of fusion proteins comprising peptides of ApoB100 (forexample SEQ ID NO: 1-302) and Cholera toxin B (CTB) were prepared andevaluated as described in WO 2011/095628, the contents of which isherein incorporated by reference in its entirety.

In some embodiments, the ApoB100 peptides described herein orcombinations thereof, or analogs, pharmaceutical equivalents and/orpeptidomimetics thereof are modified peptides. “Modified peptide” mayinclude the incorporation of lactam-bridge, head-to-tail cyclization,non-natural amino acids into the peptides of the invention, includingsynthetic non-native amino acids, substituted amino acids, or one ormore D-amino acids into the peptides (or other components of thecomposition, with exception for protease recognition sequences) isdesirable in certain situations. D-amino acid-containing peptidesexhibit increased stability in vitro or in vivo compared to L-aminoacid-containing forms. Thus, the construction of peptides incorporatingD-amino acids can be particularly useful when greater in vivo orintracellular stability is desired or required. More specifically,D-peptides are resistant to endogenous peptidases and proteases, therebyproviding better oral trans-epithelial and transdermal delivery oflinked drugs and conjugates, improved bioavailability ofmembrane-permanent complexes (see below for further discussion), andprolonged intravascular and interstitial lifetimes when such propertiesare desirable. The use of D-isomer peptides can also enhance transdermaland oral trans-epithelial delivery of linked drugs and other cargomolecules. Additionally, D-peptides cannot be processed efficiently formajor histocompatibility complex class II-restricted presentation to Thelper cells, and are therefore less likely to induce humoral immuneresponses in the whole organism. Peptide conjugates can therefore beconstructed using, for example, D-isomer forms of cell penetratingpeptide sequences, L-isomer forms of cleavage sites, and D-isomer formsof therapeutic peptides. Therefore, in some embodiments the peptides asdisclosed comprise L and D amino acids, wherein no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 D-amino acids are included. In certain aspects, thepeptides comprise more than 10 D-amino acids, and in certain aspects allthe amino acids of the peptides are D-amino acids.

In some embodiments, the ApoB100 peptides described herein orcombinations thereof, or analogs, pharmaceutical equivalents and/orpeptidomimetics thereof are retro-inverso peptides the ApoB100 peptidesdescribed herein or combinations thereof, or analogs, pharmaceuticalequivalents and/or peptidomimetics thereof. A “retro-inverso peptide”refers to a peptide with a reversal of the direction of the peptide bondon at least one position, i.e., a reversal of the amino- andcarboxy-termini with respect to the side chain of the amino acid. Thus,a retro-inverso analogue has reversed termini and reversed direction ofpeptide bonds while approximately maintaining the topology of the sidechains as in the native peptide sequence. The retro-inverso peptide cancontain L-amino acids or D-amino acids, or a mixture of L-amino acidsand D-amino acids, up to all of the amino acids being the D-isomer.Partial retro-inverso peptide analogues are polypeptides in which onlypart of the sequence is reversed and replaced with enantiomeric aminoacid residues. Since the retro-inverted portion of such an analogue hasreversed amino and carboxyl termini, the amino acid residues flankingthe retro-inverted portion are replaced by side-chain-analogousα-substituted geminal-diaminomethanes and malonates, respectively.Retro-inverso forms of cell penetrating peptides have been found to workas efficiently in translocating across a membrane as the natural forms.Synthesis of retro-inverso peptide analogues are described in Bonelli,F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A andViscomi, G. C, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S.Pat. No. 6,261,569, which are incorporated herein in their entirety byreference. Processes for the solid-phase synthesis of partialretro-inverso peptide analogues have been described (EP 97994-B) whichis also incorporated herein in its entirety by reference.

Other variants of the peptides described herein (for example, theApoB100 peptides described herein or combinations thereof, or analogs,pharmaceutical equivalents and/or peptidomimetics thereof) can compriseconservatively substituted sequences, meaning that one or more aminoacid residues of an original peptide are replaced by different residues,and that the conservatively substituted peptide retains a desiredbiological activity, i.e., the ability to treat atherosclerosis that isessentially equivalent to that of the original peptide. Examples ofconservative substitutions include substitution of amino acids that donot alter the secondary and/or tertiary structure of the ApoB100peptides described herein or combinations thereof, or analogs,pharmaceutical equivalents and/or peptidomimetics thereof, substitutionsthat do not change the overall or local hydrophobic character,substitutions that do not change the overall or local charge,substitutions by residues of equivalent side chain size, orsubstitutions by side chains with similar reactive groups.

Other examples involve substitution of amino acids that have not beenevolutionarily conserved in the parent sequence across species.Advantageously, in some embodiments, these conserved amino acids andstructures are not altered when generating conservatively substitutedsequences.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics or substitutions of residues with similarside chain volume are well known. Isolated peptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity, e.g.reducing atherosclerosis is retained, as determined by the assaysdescribed elsewhere herein.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues can be divided intogroups based on common side-chain properties: (1) hydrophobic:Norleucine, Met, Ala, Val, Leu, Ile, Phe, Trp; (2) neutral hydrophilic:Cys, Ser, Thr, Asn, Gln, Ala, Tyr, His, Pro, Gly; (3) acidic: Asp, Glu;(4) basic: His, Lys, Arg; (5) residues that influence chain orientation:Gly, Pro; (6) aromatic: Trp, Tyr, Phe, Pro, His, or hydroxyproline.Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Particularly preferred conservative substitutions for use in thevariants described herein are as follows: Ala into Gly or into Ser; Arginto Lys; Asn into Gln or into His; Asp into Glu or into Asn; Cys intoSer; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asnor into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lysinto Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Pheinto Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyror into Phe; Tyr into Phe or into Trp; and/or Phe into Val, into Tyr,into Ile or into Leu. In general, conservative substitutions encompassresidue exchanges with those of similar physicochemical properties (i.e.substitution of a hydrophobic residue for another hydrophobic aminoacid).

Any cysteine residue not involved in maintaining the proper conformationof the isolated peptide as described herein can also be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)can be added to the isolated peptide as described herein to improve itsstability or facilitate multimerization.

As used herein, a “functional fragment” is a fragment or segment of apeptide comprising at least 3, at least 4 or at least 5 amino acids andwhich can treat and/or atherosclerosis according to the assays describedherein. A functional fragment can comprise conservative substitutions ofthe sequences disclosed herein so long as they preserve the function oftreating and/or reducing atherosclerosis.

To enhance stability, bioavailability, and/or delivery of the peptidesinto the cells, the peptides can be modified. For example, in someembodiments, an isolated peptide as described herein can comprise atleast one peptide bond replacement. A single peptide bond or multiplepeptide bonds, e.g. 2 bonds, 3 bonds, 4 bonds, 5 bonds, or 6 or morebonds, or all the peptide bonds can be replaced. An isolated peptide asdescribed herein can comprise one type of peptide bond replacement ormultiple types of peptide bond replacements, e.g. 2 types, 3 types, 4types, 5 types, or more types of peptide bond replacements. Non-limitingexamples of peptide bond replacements include urea, thiourea, carbamate,sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylaceticacid, para-(aminoalkyl)-phenylacetic acid,meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronicester, olefinic group, and derivatives thereof. In some embodiments, theApoB100 peptides described herein or combinations thereof, or analogs,pharmaceutical equivalents and/or peptidomimetics thereof, areconjugated with agents that increase retention in the body. Examples ofagents that increase retention include but are not limited to cellulose,fatty acids, polyethylene glycol (PEG) or combinations thereof.

In some embodiments, an isolated peptide as described herein cancomprise naturally occurring amino acids commonly found in polypeptidesand/or proteins produced by living organisms, e.g. Ala (A), Val (V), Leu(L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr(T), Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Lys (K), Arg(R), and His (H). In some embodiments, an isolated peptide as describedherein can comprise alternative amino acids. Non-limiting examples ofalternative amino acids include, D-amino acids; beta-amino acids;homocysteine, phosphoserine, phosphothreonine, phosphotyrosine,hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine(3-mercapto-D-valine), ornithine, citruline, alpha-methyl-alanine,para-benzoylphenylalanine, para-amino phenylalanine,p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, andtert-butylglycine), diaminobutyric acid,7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine,biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline,norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid,pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylicacid, 1-amino-1-cyclohexanecarboxylic acid, amino-benzoic acid,amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine,nipecotic acid, alpha-amino butyric acid, thienyl-alanine,t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs;azide-modified amino acids; alkyne-modified amino acids; cyano-modifiedamino acids; and derivatives thereof.

In some embodiments, an isolated peptide can be modified, e.g. a moietycan be added to one or more of the amino acids comprising the peptide.In some embodiments, an isolated peptide as described herein cancomprise one or more moiety molecules, e.g. 1 or more moiety moleculesper peptide, 2 or more moiety molecules per peptide, 5 or more moietymolecules per peptide, 10 or more moiety molecules per peptide or moremoiety molecules per peptide. In some embodiments, an isolated peptideas described herein can comprise one more types of modifications and/ormoieties, e.g. 1 type of modification, 2 types of modifications, 3 typesof modifications or more types of modifications. Non-limiting examplesof modifications and/or moieties include PEGylation; glycosylation;HESylation; ELPylation; lipidation; acetylation; amidation; end-cappingmodifications; cyano groups; phosphorylation; and cyclization. In someembodiments, an end-capping modification can comprise acetylation at theN-terminus, N-terminal acylation, and N-terminal formylation. In someembodiments, an end-capping modification can comprise amidation at theC-terminus, introduction of C-terminal alcohol, aldehyde, ester, andthioester moieties.

An isolated peptide as described herein can be coupled and or connectedto a second functional molecule, peptide and/or polypeptide. In someembodiments, an isolated peptide as described herein is coupled to atargeting molecule. In some embodiments, an isolated peptide asdescribed herein is coupled to a targeting molecule by expressing thepeptide and the targeting molecule as a fusion peptide, optionally witha peptide linker sequence interposed between them. As used herein a“targeting molecule” can be any molecule, e.g. a peptide, antibody orfragment thereof, antigen, targeted liposome, or a small molecule thatcan bind to or be bound by a specific cell or tissue type. By way ofnon-limiting example, if it is desired to target an atheroscleroticregion (e.g. to treat, inhibit, reduce the severity of and/or slowprogression atherosclerosis in SLE subjects), an isolated peptidecomprising the amino acid sequence of any of SEQ ID NO: 1-302 (forexample, P210) could be coupled to an antibody or fragment thereof whichis specific for the target region.

In some embodiments, an isolated peptide as described herein can be afusion peptide or polypeptide. A fusion polypeptide can comprise apeptide linker domain interposed between the first domain of the peptidecomprising an amino acid sequence of SEQ ID NOs: 1-302 or derivatives,variants, functional fragments, prodrug, or analog thereof as describedherein and at least a second domain of the fusion peptide. The firstpeptide domain can be the N-terminal domain or the C-terminal domain oran internal sequence in the case where the partner domain forms afterfragment complementation of constituent parts. Methods of synthesizingor producing a fusion protein are well known to those of ordinary skillin the art. The term “fusion protein” as used herein refers to arecombinant protein of two or more proteins. Fusion proteins can beproduced, for example, by a nucleic acid sequence encoding one proteinis joined to the nucleic acid encoding another protein such that theyconstitute a single open-reading frame that can be translated in thecells into a single polypeptide harboring all the intended proteins. Theorder of arrangement of the proteins can vary. Fusion proteins caninclude an epitope tag or a half-life extender. Epitope tags includebiotin, FLAG tag, c-myc, hemaglutinin, His6, digoxigenin, FITC, Cy3,Cy5, green fluorescent protein, V5 epitope tags, GST, β-galactosidase,AU1, AU5, and avidin. Half-life extenders include Fc domain and serumalbumin.

In some embodiments, an isolated peptide as described herein can be apharmaceutically acceptable prodrug. As used herein, a “prodrug” refersto compounds that can be converted via some chemical or physiologicalprocess (e.g., enzymatic processes and metabolic hydrolysis) to atherapeutic agent. Thus, the term “prodrug” also refers to a precursorof a biologically active compound that is pharmaceutically acceptable. Aprodrug may be inactive when administered to a subject, i.e. an ester,but is converted in vivo to an active compound, for example, byhydrolysis to the free carboxylic acid or free hydroxyl. The prodrugcompound often offers advantages of solubility, tissue compatibility ordelayed release in an organism. The term “prodrug” is also meant toinclude any covalently bonded carriers, which release the activecompound in vivo when such prodrug is administered to a subject.Prodrugs of an active compound may be prepared by modifying functionalgroups present in the active compound in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent active compound. Prodrugs include compounds wherein ahydroxy, amino or mercapto group is bonded to any group that, when theprodrug of the active compound is administered to a subject, cleaves toform a free hydroxy, free amino or free mercapto group, respectively.Examples of prodrugs include, but are not limited to, acetate, formateand benzoate derivatives of an alcohol or acetamide, formamide andbenzamide derivatives of an amine functional group in the activecompound and the like. See Harper, “Drug Latentiation” in Jucker, ed.Progress in Drug Research 4:221-294 (1962); Morozowich et al,“Application of Physical Organic Principles to Prodrug Design” in E. B.Roche ed. Design of Biopharmaceutical Properties through Prodrugs andAnalogs, APHA Acad. Pharm. Sci. 40 (1977); Bioreversible Carriers inDrug in Drug Design, Theory and Application, E. B. Roche, ed., APHAAcad. Pharm. Sci. (1987); Design of Prodrugs, H. Bundgaard, Elsevier(1985); Wang et al. “Prodrug approaches to the improved delivery ofpeptide drug” in Curr. Pharm. Design. 5(4):265-287 (1999); Pauletti etal. (1997) Improvement in peptide bioavailability: Peptidomimetics andProdrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al.(1998) “The Use of Esters as Prodrugs for Oral Delivery of (3-Lactamantibiotics,” Pharm. Biotech. 11:345-365; Gaignault et al. (1996)“Designing Prodrugs and Bioprecursors I. Carrier Prodrugs,” Pract. Med.Chem. 671-696; Asgharnejad, “Improving Oral Drug Transport”, inTransport Processes in Pharmaceutical Systems, G. L. Amidon, P. I. Leeand E. M. Topp, Eds., Marcell Dekker, p. 185-218 (2000); Balant et al.,“Prodrugs for the improvement of drug absorption via different routes ofadministration”, Eur. J. Drug Metab. Pharmacokinet., 15(2): 143-53(1990); Balimane and Sinko, “Involvement of multiple transporters in theoral absorption of nucleoside analogues”, Adv. Drug Delivery Rev.,39(1-3): 183-209 (1999); Browne, “Fosphenytoin (Cerebyx)”, Clin.Neuropharmacol. 20(1): 1-12 (1997); Bundgaard, “Bioreversiblederivatization of drugs—principle and applicability to improve thetherapeutic effects of drugs”, Arch. Pharm. Chemi 86(1): 1-39 (1979);Bundgaard H. “Improved drug delivery by the prodrug approach”,Controlled Drug Delivery 17: 179-96 (1987); Bundgaard H. “Prodrugs as ameans to improve the delivery of peptide drugs”, Arfv. Drug DeliveryRev. 8(1): 1-38 (1992); Fleisher et al. “Improved oral drug delivery:solubility limitations overcome by the use of prodrugs”, Arfv. DrugDelivery Rev. 19(2): 115-130 (1996); Fleisher et al. “Design of prodrugsfor improved gastrointestinal absorption by intestinal enzymetargeting”, Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A): 360-81,(1985); Farquhar D, et al., “Biologically ReversiblePhosphate-Protective Groups”, Pharm. Sci., 72(3): 324-325 (1983);Freeman S, et al., “Bioreversible Protection for the Phospho Group:Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)Methylphosphonate with Carboxyesterase,” Chem. Soc., Chem. Commun.,875-877 (1991); Friis and Bundgaard, “Prodrugs of phosphates andphosphonates: Novel lipophilic alphaacyloxyalkyl ester derivatives ofphosphate- or phosphonate containing drugs masking the negative chargesof these groups”, Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangwar et al.,“Prodrug, molecular structure and percutaneous delivery”, Des. Biopharm.Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21. (1977);Nathwani and Wood, “Penicillins: a current review of their clinicalpharmacology and therapeutic use”, Drugs 45(6): 866-94 (1993); Sinhababuand Thakker, “Prodrugs of anticancer agents”, Adv. Drug Delivery Rev.19(2): 241-273 (1996); Stella et al., “Prodrugs. Do they have advantagesin clinical practice?”, Drugs 29(5): 455-73 (1985); Tan et al.“Development and optimization of anti-HIV nucleoside analogs andprodrugs: A review of their cellular pharmacology, structure-activityrelationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1-3):117-151 (1999); Taylor, “Improved passive oral drug delivery viaprodrugs”, Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino andBorchardt, “Prodrug strategies to enhance the intestinal absorption ofpeptides”, Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus,“Concepts for the design of anti-HIV nucleoside prodrugs for treatingcephalic HIV infection”, Adv. Drug Delivery Rev.: 39(1-3):63-80 (1999);Waller et al., “Prodrugs”, Br. J. Clin. Pharmac. 28: 497-507 (1989),which are incorporated by reference herein in their entireties.

In some embodiments, an isolated peptide as described herein can be apharmaceutically acceptable solvate. The term “solvate” refers to anisolated peptide as described herein in the solid state, whereinmolecules of a suitable solvent are incorporated in the crystal lattice.A suitable solvent for therapeutic administration is physiologicallytolerable at the dosage administered. Examples of suitable solvents fortherapeutic administration are ethanol and water. When water is thesolvent, the solvate is referred to as a hydrate. In general, solvatesare formed by dissolving the compound in the appropriate solvent andisolating the solvate by cooling or using an antisolvent. The solvate istypically dried or azeotroped under ambient conditions.

In some embodiments, an isolated peptide as described herein can be in anon-crystalline, i.e. amorphous solid form.

In one aspect, described herein is a vector comprising a nucleic acidencoding a peptide as described herein. The term “vector”, as usedherein, refers to a nucleic acid construct designed for delivery to ahost cell or for transfer between different host cells. As used herein,a vector can be viral or non-viral. The term “vector” encompasses anygenetic element that is capable of replication when associated with theproper control elements and that can transfer gene sequences to cells. Avector can include, but is not limited to, a cloning vector, anexpression vector, a plasmid, phage, transposon, cosmid, chromosome,virus, virion, etc. Many vectors useful for transferring exogenous genesinto target mammalian cells are available. The vectors can be episomal,e.g., plasmids, virus derived vectors such cytomegalovirus, adenovirus,etc., or can be integrated into the target cell genome, throughhomologous recombination or random integration, e.g., retrovirus derivedvectors such MMLV, HIV-1, ALV, etc. Many viral vectors are known in theart and can be used as carriers of a nucleic acid modulatory compoundinto the cell. For example, constructs containing the nucleic acidencoding a polypeptide can be integrated and packaged intonon-replicating, defective viral genomes like Adenovirus,Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others,including retroviral and lentiviral vectors, for infection ortransduction into cells. Alternatively, the construct can beincorporated into vectors capable of episomal replication, e.g. EPV andEBV vectors. The nucleic acid incorporated into the vector can beoperatively linked to an expression control sequence such that theexpression control sequence controls and regulates the transcription andtranslation of that polynucleotide sequence.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector can comprise additional elements, for example, theexpression vector can have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification.

The term “transfection” as used herein to methods, such as chemicalmethods, to introduce exogenous nucleic acids, such as the nucleic acidsequences encoding a peptide as described herein into a cell. As usedherein, the term transfection does not encompass viral-based methods ofintroducing exogenous nucleic acids into a cell. Methods of transfectioninclude physical treatments (electroporation, nanoparticles,magnetofection), and chemical-based transfection methods. Chemical-basedtransfection methods include, but are not limited to those that usecyclodextrin, polymers, liposomes, nanoparticles, cationic lipids ormixtures thereof (e.g., DOPA, Lipofectamine and UptiFectin), andcationic polymers, such as DEAE-dextran or polyethylenimine.

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the nucleic acid encoding a peptide as described herein inplace of non-essential viral genes. The vector and/or particle can beutilized for the purpose of transferring any nucleic acids into cellseither in vitro or in vivo. Numerous forms of viral vectors are known inthe art. The term “replication incompetent” when used in reference to aviral vector means the viral vector cannot further replicate and packageits genomes. For example, when the cells of a subject are infected withreplication incompetent recombinant adeno-associated virus (rAAV)virions, the heterologous (also known as transgene) gene is expressed inthe patient's cells, but, the rAAV is replication defective (e.g., lacksaccessory genes that encode essential proteins for packaging the virus)and viral particles cannot be formed in the patient's cells. The term“transduction” as used herein refers to the use of viral particles orviruses to introduce exogenous nucleic acids into a cell.

Retroviruses, such as lentiviruses, provide a convenient platform fordelivery of nucleic acid sequences encoding an agent of interest. Aselected nucleic acid sequence can be inserted into a vector andpackaged in retroviral particles using techniques known in the art. Therecombinant virus can then be isolated and delivered to cells, e.g. invitro or ex vivo. Retroviral systems are well known in the art and aredescribed in, for example, U.S. Pat. No. 5,219,740; Kurth and Bannert(2010) “Retroviruses: Molecular Biology, Genomics and Pathogenesis”Calster Academic Press (ISBN:978-1-90455-55-4); and Hu and PathakPharmacological Reviews 2000 52:493-512; which are incorporated byreference herein in their entirety.

In some embodiments, a nucleotide sequence of interest is inserted intoan adenovirus-based expression vector. Unlike retroviruses, whichintegrate into the host genome, adenoviruses persist extrachromosomallythus minimizing the risks associated with insertional mutagenesis(Haj-Ahmad and Graham (1986) J. Virol. 57:267-74; Bett et al. (1993) J.Virol. 67:5911-21; Mittereder et al. (1994) Human Gene Therapy 5:717-29;Seth et al. (1994) J. Virol. 68:933-40; Barr et al. (1994) Gene Therapy1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-29; and Rich et al.(1993) Human Gene Therapy 4:461-76). Adenoviral vectors have severaladvantages in gene therapy. They infect a wide variety of cells, have abroad host-range, exhibit high efficiencies of infectivity, directexpression of heterologous sequences at high levels, and achievelong-term expression of those sequences in vivo. The virus is fullyinfective as a cell-free virion so injection of producer cell lines isnot necessary. With regard to safety, adenovirus is not associated withsevere human pathology, and the recombinant vectors derived from thevirus can be rendered replication defective by deletions in theearly-region 1 (“E1”) of the viral genome. Adenovirus can also beproduced in large quantities with relative ease. For all these reasonsvectors derived from human adenoviruses, in which at least the E1 regionhas been deleted and replaced by a gene of interest, have been usedextensively for gene therapy experiments in the pre-clinical andclinical phase. Adenoviral vectors for use with the compositions andmethods described herein can be derived from any of the variousadenoviral serotypes, including, without limitation, any of the over 40serotype strains of adenovirus, such as serotypes 2, 5, 12, 40, and 41.The adenoviral vectors of used in the methods described herein aregenerally replication-deficient and contain the sequence of interestunder the control of a suitable promoter. For example, U.S. Pat. No.6,048,551, incorporated herein by reference in its entirety, describesreplication-deficient adenoviral vectors that include a human gene underthe control of the Rous Sarcoma Virus (RSV) promoter. Other recombinantadenoviruses of various serotypes, and comprising different promotersystems, can be created by those skilled in the art. See, e.g., U.S.Pat. No. 6,306,652, incorporated herein by reference in its entirety.Other useful adenovirus-based vectors for delivery of nucleic acidsequences include, but are not limited to: “minimal” adenovirus vectorsas described in U.S. Pat. No. 6,306,652, which retain at least a portionof the viral genome required for encapsidation (the encapsidationsignal), as well as at least one copy of at least a functional part or aderivative of the ITR; and the “gutless” (helper-dependent) adenovirusin which the vast majority of the viral genome has been removed andwhich produce essentially no viral proteins, such vectors can permitgene expression to persist for over a year after a single administration(Wu et al. (2001) Anesthes. 94:1119-32; Parks (2000) Clin. Genet.58:1-11; Tsai et al. (2000) Curr. Opin. Mol. Ther. 2:515-23).

In some embodiments, a nucleotide sequence encoding a peptide asdescribed herein is inserted into an adeno-associated virus-basedexpression vector. AAV is a parvovirus which belongs to the genusDependovirus and has several features not found in other viruses. AAVcan infect a wide range of host cells, including non-dividing cells. AAVcan infect cells from different species. AAV has not been associatedwith any human or animal disease and does not appear to alter thebiological properties of the host cell upon integration. Indeed, it isestimated that 80-85% of the human population has been exposed to thevirus. Finally, AAV is stable at a wide range of physical and chemicalconditions, facilitating production, storage and transportation. AAV isa helper-dependent virus; that is, it requires co-infection with ahelper virus (e.g., adenovirus, herpesvirus or vaccinia) in order toform AAV virions in the wild. In the absence of co-infection with ahelper virus, AAV establishes a latent state in which the viral genomeinserts into a host cell chromosome, but infectious virions are notproduced. Subsequent infection by a helper virus rescues the integratedgenome, allowing it to replicate and package its genome into infectiousAAV virions. While AAV can infect cells from different species, thehelper virus must be of the same species as the host cell. Thus, forexample, human AAV will replicate in canine cells co-infected with acanine adenovirus. Adeno-associated virus (AAV) has been used withsuccess in gene therapy. AAV has been engineered to deliver genes ofinterest by deleting the internal nonrepeating portion of the AAV genome(i.e., the rep and cap genes) and inserting a heterologous sequence (inthis case, the sequence encoding the agent) between the ITRs. Theheterologous sequence is typically functionally linked to a heterologouspromoter (constitutive, cell-specific, or inducible) capable of drivingexpression in the patient's target cells under appropriate conditions.Recombinant AAV virions comprising a nucleic acid sequence encoding anagent of interest can be produced using a variety of art-recognizedtechniques, as described in U.S. Pat. Nos. 5,139,941; 5,622,856;5,139,941; 6,001,650; and 6,004,797, the contents of each of which areincorporated by reference herein in their entireties. Vectors and celllines necessary for preparing helper virus-free rAAV stocks arecommercially available as the AAV Helper-Free System (Catalog No.240071) (Agilent Technologies, Santa Clara, Calif.).

Additional viral vectors useful for delivering nucleic acid moleculesencoding a peptide as described herein include those derived from thepox family of viruses, including vaccinia virus and avian poxvirus.Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can be used to deliver the genes. The use of avipox vectors in cells ofhuman and other mammalian species is advantageous with regard to safetybecause members of the avipox genus can only productively replicate insusceptible avian species. Methods for producing recombinantavipoxviruses are known in the art and employ genetic recombination,see, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectors,can also be used for delivery of sequence encoding a peptide asdescribed herein (Michael et al. (1993) J. Biol. Chem. 268:6866-69 andWagner et al. (1992) Proc. Natl. Acad. Sci. USA 89:6099-6103). Membersof the Alphavirus genus, for example the Sindbis and Semliki Forestviruses, can also be used as viral vectors for delivering a nucleic acidsequence (See, e.g., Dubensky et al. (1996) J. Virol. 70:508-19; WO95/07995; WO 96/17072).

In some embodiments, the vector further comprises a signal peptideoperably linked to the peptide. Signal peptides are terminally (usuallyN-terminally) located peptide sequences that provide for passage of theprotein into or through a membrane. Different signal peptides can be ofuse in different applications. For example, as regards a cellular systemfor the production of isolated peptides as described herein, a secretorysignal peptide can permit increased yields and ease of purification. Asa further example, as regards cells which produce peptides as describedherein and which are administered for therapeutic purposes to a subject,multiple signal peptides, e.g. a peptide signaling for secretion fromthe first cell, a peptide signaling for internalization by a secondcell, and a final peptide signaling for nuclear localization canincrease the amount of peptide reaching the target environment. As afurther example, as regards, e.g. gene therapy applications, a peptidesignaling for nuclear localization can increase the amount of peptidereaching the target environment. Signal peptides are known in the art.Non-limiting examples of nuclear localization signal (NLS) peptides foruse in mammalian cells include; the SV40 large T-antigen NLS (PKKKRKV)(SEQ ID NO: 305); the nucleoplasmin NLS (KR[PAATKKAGQA]KKKK)(SEQ ID NO:306); the K-K/R-X-K/R (SEQ ID NO: 307) consensus NLS (KKXR (SEQ ID NO:308); KKXK (SEQ ID NO: 309); KRXK (SEQ ID NO: 310); KRXR (SEQ ID NO:311); and PY-NLSs (see, e.g. Dingwall et al. J Cell Biol 188 107:841-9and Makkerh et al. Curr Biol. 1996 6:1025-7; both of which areincorporated by reference herein in their entireties, for furtherdiscussion). Non-limiting examples of secretion signal peptides for usein mammalian cells include human albumin signal peptide(MKWVTFISLLFLFSSAYS) (SEQ ID NO: 312); human chymotrypsin signal peptide(MAFLWLLSCWALLGTTGF) (SEQ ID NO: 313); human interleukin-2 signalpeptide (MQLLSCIALILALV) (SEQ ID NO: 314); human trypsinogen-2 signalpeptide (MNLLLILTFVAAAVA) (SEQ ID NO: 315); and sequences which includea coding region for a signal for precursor cleavage by signal peptidase,furin or other prohormone convertases (e.g., PC3). For example, a signal(peptide) sequence which is cleaved by furin (also known as PACE, seeU.S. Pat. No. 5,460,950), other subtilisins (including PC2, PC1/PC3,PACE4, PC4, PC5/PC6, LPC/PC7IPC8/SPC7 and SKI-I; Nakayama, Biochem. J.,327:625-635 (1997)); enterokinase (see U.S. Pat. No. 5,270,181) orchymotrypsin can be introduced into the signal (peptide) sequence asdefined herein. Additional signal peptides are known in the art and thechoice of signal peptide can be influenced by the cell type, growthconditions, and the desired destination of the peptide.

In one aspect, described herein is a cell expressing a vector comprisinga nucleic acid encoding a peptide as described herein. In someembodiments, the cell expressing a vector as described herein is a cellsuitable for the production of polypeptides. A cell suitable for theproduction of polypeptides can be a prokaryotic or eukaryotic cell, e.g.bacteria, virus, yeast, fungi, mammalian cells, insect cells, plantcells, and the like. By way of non-limiting example, cells for theproduction of proteins are commercially available, e.g. bacterial cells(BL21 derived cells—Cat. No. 60401-1, Lucigen; Middleton, Wis. andmammalian cells (293 F cells—Cat. No. 11625-019, Invitrogen; GrandIsland, N.Y.).

Recombinant molecules, e.g. vectors as described herein, can beintroduced into cells via transformation, particularly transduction,conjugation, lipofection, protoplast fusion, mobilization, particlebombardment, electroporation (Neumann et al., “Gene Transfer into MouseLyoma Cells by Electroporation in High Electric Fields,” EMBO J.1(7):841-845 (1982); Wong et al., “Electric Field Mediated GeneTransfer,” Biochem Biophys Res Commun 107(2):584-587 (1982); Potter etal., “Enhancer-dependent Expression of Human Kappa Immunoglobulin GenesIntroduced into Mouse pre-B Lymphocytes by Electroporation,” Proc. Natl.Acad. Sci. USA 81(22):7161-7165 (1984), which are hereby incorporated byreference in their entirety), polyethylene glycol-mediated DNA uptake(Joseph Sambrook & David W. Russell, Molecular Cloning: A LaboratoryManual cp. 16 (2d ed. 1989), which is hereby incorporated by referencein its entirety), or fusion of protoplasts with other entities (e.g.,minicells, cells, lysosomes, or other fusible lipid-surfaced bodies thatcontain the chimeric gene) (Fraley et al., “Liposome-mediated Deliveryof Tobacco Mosaic Virus RNA into Tobacco Protoplasts: A Sensitive Assayfor Monitoring Liposome-protoplast Interactions,” Proc. Natl. Acad. Sci.USA, 79(6):1859-1863 (1982), which is hereby incorporated by referencein its entirety). The host cell is then cultured in a suitable medium,and under conditions suitable for expression of the protein orpolypeptide of interest. After cultivation, the cell is disrupted byphysical or chemical means, and the protein or polypeptide purified fromthe resultant crude extract. Alternatively, cultivation may includeconditions in which the protein or polypeptide is secreted into thegrowth medium of the recombinant host cell, and the protein orpolypeptide is isolated from the growth medium. Alternative methods maybe used as suitable.

The terms “enhancer” and “enhance” as it pertains to a molecule inconnection with CD8 T cell refers to the ability of a molecule to modifythe immune response by promoting the activation of cells of the immunesystem. The choice of appropriate enhancer can allow control ofactivation of the immune response. Exemplary enhancers include cytokinessuch as IL-2. The term “cytokine” as used herein refers cell signalingmolecules that act as has immunomodulating agents, and comprise proteinssuch as interleukins and interferons as would be identifiable to askilled person. Selection of a suitable cytokine can result underappropriate conditions in the preferential induction of a humoral orcellular immune response.

In an embodiment, the enhancer can be Interleukin 2 (IL2), Interleukin15 (IL-15), TGF-beta (TGF-β), IL2-antiIL-2 antibody complex and/oradditional enhancer identifiable by a skilled person upon reading of thepresent disclosure. Reference is made to the references Mitchell et al2010 (38), Perret et al 2008 (39) and Kamimura et al 2007 (40), eachincorporated by reference in its entirety, which describe exemplary useof enhancer in connection with T cell activation.

In particular in some embodiments, the enhancing is performed byreducing CD86 expression and/or IL12 secretion by dendritic cells in theindividual.

The peptides can also be attached to adjuvants. The term “adjuvant”refers to a compound or mixture that enhances the immune response and/orpromotes the proper rate of absorption following inoculation, and, asused herein, encompasses any uptake-facilitating agent. Non-limitingexamples of adjuvants include, chemokines (e.g., defensins, HCC-1, HCC4,MCP-1, MCP-3, MCP4, MIP-1α, MIP-1β, MIP-1β, MIP-3α, RANTES); otherligands of chemokine receptors (e.g., CCR1, CCR-2, CCR-5, CCR6, CXCR-1);cytokines (e.g., IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-13, IL-15, IL-17 (A-F), IL-18; IFNα, IFN-γ; TNF-α; GM-CSF);TGF)-β; FLT-3 ligand; CD40 ligand; other ligands of receptors for thosecytokines; Th1 cytokines including, without limitation, IFN-γ, IL-12,IL-18, and TNF; Th2 cytokines including, without limitation, IL-4, IL-5,IL-10, and IL-13; and Th17 cytokines including, without limitation,IL-17 (A through F), IL-23, TGF-β and IL-6; immunostimulatory CpG motifsin bacterial DNA or oligonucleotides; derivatives of lipopolysaccharidessuch as monophosphoryl lipid A (MPL); muramyl dipeptide (MDP) andderivatives thereof (e.g., murabutide, threonyl-MDP, muramyl tripeptide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alani-ne-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE)); MF59 (see Int'l Publication No. WO90/14837); poly[di(carboxylatophenoxy)phosphazene] (PCPP polymer; VirusResearch Institute, USA); RIBI (GSK), which contains three componentsextracted from bacteria, monophosphoryl lipid A, trehalose dimycolateand cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion;OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA,Meyrin, Switzerland); heat shock proteins and derivatives thereof;Leishmania homologs of elF4a and derivatives thereof; bacterialADP-ribosylating exotoxins and derivatives thereof (e.g., geneticmutants, A and/or B subunit-containing fragments, chemically toxoidedversions); chemical conjugates or genetic recombinants containingbacterial ADP-ribosylating exotoxins or derivatives thereof; C3d tandemarray; lipid A and derivatives thereof (e.g., monophosphoryl ordiphosphoryl lipid A, lipid A analogs, AGP, AS02, AS04, DC-Chol, Detox,OM-174); ISCOMS and saponins (e.g., Quil A, QS-21, STIMULON® (CambridgeBioscience, Worcester, Mass.)); squalene; superantigens; or salts (e.g.,aluminum hydroxide or phosphate, calcium phosphate). See also Nohria etal. Biotherapy, 7:261-269, 1994; Richards et al., in Vaccine Design,Eds. Powell et al., Plenum Press, 1995; and Pashine et al., NatureMedicine, 11:S63-S68, 4/2005) for other useful adjuvants. Furtherexamples of adjuvants can include the RIBI adjuvant system (Ribi Inc.,Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel,oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund'scomplete and incomplete adjuvants, Block co-polymer (CytRx, AtlantaGa.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), and SAF-M(Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A orother saponin fraction, monophosphoryl lipid A, and Avridine lipid-amineadjuvant, and METASTIM®. Other suitable adjuvants can include, forexample, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil or hydrocarbon emulsions, keyholelimpet hemocyanins, dinitrophenol, and others.

In some embodiment, cell may be genetically engineered to express thepeptides described herein and the genetically engineered cells may beused for cell therapy. Examples of cells that may be used include butare not limited to, dendritic cells, T-lymphocytes (T-cells), naïve Tcells (T_(N)), memory T cells (for example, central memory T cells(T_(CM)), effector memory cells (T_(EM))), natural killer cells,hematopoietic stem cells and/or pluripotent embryonic/induced stem cellscapable of giving rise to therapeutically relevant progeny. In anembodiment, the genetically engineered cells are autologous cells. Byway of example, individual T-cells of the invention may be CD4+/CD8−,CD4−/CD8+, CD4−/CD8− or CD4+/CD8+. The T-cells may be a mixed populationof CD4+/CD8− and CD4−/CD8+ cells or a population of a single clone. CD4+T-cells may produce IL-2, IFNγ, TNFα and other T-cell effector cytokineswhen co-cultured in vitro with cells expressing the peptides (forexample CD20+ and/or CD19+ tumor cells). CD8⁺ T-cells may lyseantigen-specific target cells when co-cultured in vitro with the targetcells. In some embodiments, T cells may be any one or more ofCD45RA⁺CD62L⁺ naïve cells, CD45RO⁺CD62L⁺ central memory cells, CD62L⁻effector memory cells or a combination thereof (Berger et al., Adoptivetransfer of virus-specific and tumor-specific T cell immunity. Curr OpinImmunol 2009 21(2)224-232).

In some embodiments, tolerized antigen presenting cells may be used incell therapy. Examples include B cells, dendritic cells, macrophages andthe like. The cells may be of any origin, including from humans. Thecells may be tolerized using the peptides described herein. In someembodiments, the cells are tolerized in the presence of cytokines.

In some embodiments, the cell producing the peptide as described hereincan be administered to a subject, e.g. for treating, inhibiting,reducing the severity of and/or slow progression of atherosclerosis(such as accelerated atherosclerosis) in subjects with SLE.

In some embodiments, nanoparticles containing the peptide as describedherein can be administrated to a subject. In some embodiments, thenanoparticles for use with the peptides described herein may be asdescribed in Levine et al., Polymersomes: A new multi-functional toolfor cancer diagnosis and therapy. Methods 2008 Vol 46 pg 25-32 or asdescribed in S Jain, et al., Gold nanoparticles as novel agents forcancer therapy. Br J Radiol. 2012 February; 85(1010): 101-113.

In some embodiments, the cell expressing a vector encoding a peptide asdescribed herein can be a cell of a subject, e.g. a subject administeredgene therapy for the treatment, inhibition, reduction of severity and/orslow progression of atherosclerosis (such as acceleratedatherosclerosis) in subjects with SLE. Vectors for gene therapy cancomprise viral or non-viral vectors as described elsewhere herein.

Some embodiments of the present invention can be defined as any of thefollowing numbered paragraphs:

-   1. A method of treating, inhibiting, preventing, reducing the    severity of slow progression of and/or promoting prophylaxis of    cardiovascular diseases in subjects with SLE comprising: (a)    providing a composition comprising one or more peptides of ApoB or    derivatives, pharmaceutical equivalents, peptidomimetics or analogs    thereof; and (b) administering an effective amount of the    composition to the subject, so as to treat, inhibit, prevent, reduce    the severity of, slow progression of and/or promote prophylaxis of    cardiovascular diseases in subjects with (SLE).-   2. A method of treating, inhibiting, preventing, reducing the    severity of, slowing progression of, and/or promoting prophylaxis of    systemic lupus erythematosus (SLE) in subjects in need thereof    comprising: (a) providing a composition comprising one or more    peptides of ApoB100 (“ApoB”) or derivatives, pharmaceutical    equivalents, peptidomimetics or analogs thereof; and (b)    administering an effective amount of the composition to the subject,    so as to treat, inhibit, prevent, reduce the severity, slow the    progression, and/or promote prophylaxis of SLE in the subject.-   3. The method of paragraphs 1 or 2, wherein the peptide of ApoB is    any one or more of peptides 1 to 302 of ApoB100 as set forth in SEQ    ID NO: 1 to SEQ ID NO: 302.-   4. The method of paragraph 3, wherein the peptide of ApoB100 is P210    (SEQ ID NO: 210).-   5. The method of paragraph 3, wherein the peptide of ApoB100 is P45    (SEQ ID NO: 45).-   6. The method of paragraph 3, wherein the peptide is fused to    cholera toxin B (CTB).-   7. A method of treating, inhibiting, preventing, reducing the    severity of, slowing progression of and/or promoting prophylaxis of    SLE in a subject in need thereof comprising: (a) providing a    composition comprising CD8+ T cells activated with one or more    peptides of ApoB or derivatives, pharmaceutical equivalents,    peptidomimetics or analogs thereof; and (b) administering an    effective amount of the composition to the subject so as to treat,    inhibit, prevent, reduce the severity of, slow progression of and/or    promote prophylaxis of SLE in the subject.-   8. A method of treating, inhibiting, preventing, reducing the    severity of, slowing progression of and/or promoting prophylaxis of    cardiovascular diseases in subjects with SLE comprising: (a)    providing a composition comprising CD8+ T cells activated with one    or more peptides of ApoB or derivatives, pharmaceutical equivalents,    peptidomimetics or analogs thereof; and (b) administering an    effective amount of the composition to the subject, so as to treat,    inhibit, prevent, reduce the severity of, slow progression of and/or    promote prophylaxis of cardiovascular diseases in subjects with    (SLE).-   9. The method of paragraphs 7 or 8, wherein the peptide of ApoB is    any one or more of peptides 1 to 302 of ApoB as set forth in SEQ ID    NO: 1 to SEQ ID NO: 302.-   10. The method of paragraph 9, wherein the peptide of ApoB is P210    (SEQ ID NO: 210).-   11. The method of paragraph 9, wherein the peptide of ApoB is P45    (SEQ ID NO: 45).-   12. The method of paragraphs 7 or 8, wherein the peptide is fused to    cholera toxin B (CTB).-   13. The method of paragraphs 7 or 8, wherein the method further    comprises administering an effective amount of one or more    enhancers.-   14. The method of paragraph 13, wherein the enhancers are any one or    more of IL-2, IL-10, IL-15. TGF-β, IL2/Anti-IL-2 antibody complex,    or combinations thereof.-   15. A method for treating SLE in a subject in need thereof by    passive immunization, comprising providing an antibody that binds at    least one oxidized fragment of ApoB and administering a    therapeutically or prophylactically effective amount of the    antibody, so as to treat SLE.-   16. The method of paragraph 15, wherein the antibody is a human    antibody.-   17. The method of paragraph 16, wherein the antibody comprises a    variable heavy region (V_(H)) and a variable light region (V_(L)),    wherein the V_(H) region consists of the sequence set forth in SEQ    ID NO 303 and the variable light region (V_(L)) No. 304.-   18. The method of paragraph 1, 2, 5 or 6, further comprising    administering an effective amount of an antibody that binds at least    one oxidized fragment of ApoB.-   19. The method of paragraph 18, wherein the antibody is a human    antibody.-   20. The method of paragraph 19, wherein the antibody comprises a    variable heavy region (V_(H)) and a variable light region (V_(L)),    wherein the V_(H) region consists of the sequence set forth in SEQ    ID NO 303 and the variable light region (V_(L)) No. 304.-   21. The method of paragraph 1 or 8, wherein the cardiovascular    disease is atherosclerosis.-   22. The method of paragraph 21, wherein atherosclerosis is    accelerated atherosclerosis.-   23. An assay for diagnosing SLE in a subject in need thereof    comprising:    -   Obtaining a sample from the subject;    -   Assaying the sample to determine the level of autoantibodies        against ApoB100; and    -   Determining that the subject has increased likelihood of having        SLE if the level of the autoantibodies is decreased relative to        a reference value, or determining that the subject has decreased        likelihood of having SLE if the level of autoantibodies is        increased relative to a reference value.-   24. An assay for determining likelihood of cardiovascular disease in    a subject having or suspected of having SLE comprising:    -   Obtaining a sample from the subject;    -   Assaying the sample to determine the level of autoantibodies        against ApoB100; and    -   Determining that the subject has increased likelihood of        cardiovascular disease if the level of the autoantibodies is        decreased relative to a reference value, or determining that the        subject has decreased likelihood of cardiovascular disease if        the level of autoantibodies is increased relative to a reference        value.-   25. The assay of paragraphs 23 or 24, wherein the assay comprises    using is an immunoassays.-   26. The assay of paragraph 25, wherein the immunoassay is any one or    more of ELISA, RIA, Western blotting, Southern blotting, or    combinations thereof.-   27. The assay of paragraphs 23 or 24, wherein the sample is blood,    plasma, urine, tissue or combinations thereof.-   28. The assay of paragraph 27, wherein the sample is obtained    before, during or after treatment for SLE.-   29. The assay of paragraphs 23 or 24, wherein the subject is human.-   30. The assay of paragraphs 23 or 24, wherein the reference value is    the mean or median level of autoantibodies against ApoB100 in a    population of subjects that do not have SLE.-   31. The assay of paragraphs 23 or 24, wherein the reference value is    the mean or median level of autoantibodies against ApoB100 in a    sample obtained from the subject at a different time point.-   32. The assay of paragraphs 23 or 24, wherein the reference value is    the mean or median level of autoantibodies against ApoB100 in a    population of subjects that have SLE and have undergone or are    undergoing treatment for SLE.-   33. The assay of paragraphs 23 or 24, wherein the reference value is    the mean or median level of autoantibodies against ApoB100 in a    population of subjects that have SLE and have undergone or are    undergoing treatment for SLE and have not undergone or are not    undergoing treatment for cardiovascular diseases.-   34. The assay of paragraph 24, wherein the cardiovascular disease is    atherosclerosis.-   35. The assay of paragraph 34, wherein atherosclerosis is    accelerated atherosclerosis.-   36. The assay of paragraph 23, further comprising determine the    level of soluble forms of the apoptosis-signaling receptors Fas,    TNF-R1, and/or TRAIL-R2; and determining that the subject has    increased likelihood of having SLE if the level of the said    apoptosis-signaling receptors is increased relative to a reference    value, or determining that the subject has decreased likelihood of    having SLE if the level of said apoptosis-signaling receptors is    decreased relative to a reference value.-   37. An assay for determining the efficacy of treatment for SLE in a    subject in need thereof comprising:    -   Obtaining a sample from the subject;    -   Assaying the sample to determine the level of autoantibodies        against ApoB100; and    -   Determining that the treatment is effective if the level of the        autoantibodies is increased relative to a reference value, or        determining that the treatment is ineffective if the level of        autoantibodies is decreases relative to a reference value.-   38. An assay for determining the efficacy of treatment for    cardiovascular diseases in subject with SLE comprising:    -   Obtaining a sample from the subject;    -   Assaying the sample to determine the level of autoantibodies        against ApoB100; and    -   Determining that the treatment is effective if the level of the        autoantibodies is increased relative to a reference value, or        determining that the treatment is ineffective if the level of        autoantibodies is decreases relative to a reference value.-   39. An assay for diagnosing SLE in a subject in need thereof    comprising:    -   Obtaining a sample from the subject;    -   Assaying the sample to determine the level of soluble forms of        the apoptosis-signaling receptors Fas, TNF-R1, and/or TRAIL-R2;        and    -   Determining that the subject has increased likelihood of having        SLE if the level of the said apoptosis-signaling receptors is        increased relative to a reference value, or determining that the        subject has decreased likelihood of having SLE if the level of        said apoptosis-signaling receptors is decreased relative to a        reference value.-   40. The assay of paragraph 39, further comprising assaying the    sample to determine the level of autoantibodies against ApoB100; and    determining that the subject has increased likelihood of having SLE    if the level of the autoantibodies is decreased relative to a    reference value, or determining that the subject has decreased    likelihood of having SLE if the level of autoantibodies is increased    relative to a reference value.-   41. An assay for determining likelihood of cardiovascular disease in    a subject having or suspected of having SLE comprising:    -   Obtaining a sample from the subject;    -   Assaying the sample to determine the level of soluble forms of        the apoptosis-signaling receptors Fas, TNF-R1, and/or TRAIL-R2;        and    -   Determining that the subject has increased likelihood of        cardiovascular disease if the level of said apoptosis-signaling        receptors is increased relative to a reference value, or        determining that the subject has decreased likelihood of        cardiovascular disease if the level of said apoptosis-signaling        receptors is decreased relative to a reference value.        Further Methods and Systems

Various embodiments provide a method of treating, reducing the severityof SLE in a subject, or of atherosclerosis in a subject with SLE, byadministering to the subject an antibody or antibody fragment that bindsto at least one fragment of apolipoprotein B100 (apoB100). In variousembodiments, the method provides treatment of SLE in a subject, or ofatherosclerosis in a subject with SLE, by passive immunization. Furtherembodiments provide the atherosclerosis is accelerated atherosclerosis,with which a patient presents earlier, and may develop more quickly,atherosclerotic lesions, compared to non-diseased age matched controls.

Various embodiments provide a method of reducing the likelihood ofhaving SLE, or having atherosclerosis in a subject with SLE, byadministering to the subject an antibody or antibody fragment that bindsto at least one fragment of apolipoprotein B100 (apoB100). In variousembodiments, the method provides passive immunity to the subject toreduce the likelihood of having SLE.

Various embodiments provide the antibody or antibody fragment in themethods disclosed herein binds to a native and/or an oxidized epitopeP45 of apoB100. Various embodiments provide the antibody or antibodyfragment in the methods disclosed herein only binds to a native and/oran oxidized epitope P45 of apoB100. P45 of apoB100 has a polypeptidesequence of IEIGLEGKGFEPTLEALFGK (SEQ ID NO: 45). An oxidized epitope oroxidized lipoprotein includes but is not limited to a modification onthe epitope or lipoprotein to carry malone-di-aldehyde (MDA) groups onlysines and histidines, a modification that is induced by oxidation bycopper (e.g., CuOxLDL), a modification to carry hydroxynonenal, or amodification to carry a hapten of an aldehyde. Another embodimentprovides the antibody or antibody fragment in the method disclosedherein further binds one or more fragments of apoB100.

Various embodiments provide that the method of treating or reducing theseverity of SLE in a subject, or of atherosclerosis in a subject withSLE, in a subject includes but is not limited to administering orticumabor a variant of orticumab that has identical heavy chain and/or lightchain to those of orticumab, or identical complementarity determiningregions to those of orticumab.

Various embodiments provide that the method of reducing the likelihoodof SLE in a subject, or of atherosclerosis in a subject with SLE, in asubject includes but is not limited to administering orticumab or avariant of orticumab that has identical heavy chain and/or light chainto those of orticumab, or identical complementarity determining regionsto those of orticumab. In further aspect, the method provides passiveimmunity to the subject to reduce the likelihood of having SLE.

The complementarity determining regions (CDRs) of orticumab aredescribed above in relation to FIG. 21B and FIG. 21C. The heavy chaincomplementarity determining region (HCDR) 1 (HCDR1), 2 (HCDR2) and 3(HCDR3) are set forth in SEQ ID Nos: 318, 319 and 320, respectively; andlight chain complementarity determining regions (LCDR) 1 (LCDR1), 2(LCDR2) and 3 (LCDR3) are set forth in SEQ ID Nos: 321, 322 and 323,respectively. Orticumab contains a variable heavy region (V_(H)) aminoacid sequence of SEQ ID No: 324, and a variable light region (V_(L))amino acid sequence of SEQ ID No: 325. Orticumab contains a heavy chainamino acid sequence of SEQ ID No: 316, a light chain amino acid sequenceof SEQ ID No: 317, as shown in FIG. 21B and FIG. 21C.

HCDR1, i.e., SEQ ID NO: 318, is: FSNAWMSWVRQAPG.HCDR2, i.e., SEQ ID NO: 319, is: SSISVGGHRTYYADSVKGR.HCDR3, i.e., SEQ ID NO: 320, is: ARIRVGPSGGAFDY.LCDR1, i.e., SEQ ID NO: 321, is: CSGSNTNIGKNYVS.LCDR2, i.e., SEQ ID NO: 322, is: ANSNRPS.LCDR3, i.e., SEQ ID NO: 323, is: CASWDASLNGWV.

Variable heavy region (V_(H)), i.e., SEQ ID NO:324, is as shown:

EVQLLESGGG LVQPGGSLRL SCAASGFTFS NAWMSWVRQAPGKGLEWVSS ISVGGHRTYY ADSVKGRSTI SRDNSKNTLYLQMNSLRAED TAVYYCARIR VGPSGGAFDY WGQGTLVTVS.

Variable light region (V_(L)), i.e., SEQ ID NO: 325, is as shown:

QSVLTQPPSA SGTPGQRVTI SCSGSNTNIG KNYVSWYQQLPGTAPKLLIY ANSNRPSGVP DRFSGSKSGT SASLAISGLRSEDEADYYCA SWDASLNGWV FGGGTKLTVL.

Methods are provided of treating or reducing the severity of SLE in asubject, or of atherosclerosis in a subject with SLE, in a subjectincluding administering to the subject an effective amount of anantibody or antibody fragment that binds a fragment set forth in SEQ IDNO: 45 of apoB100, and the antibody contains one or more of HCDR1,HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID Nos.:318-323, respectively.

Methods of treating or reducing the severity of SLE in a subject, or ofatherosclerosis in a subject with SLE, in a subject are also providedincluding administering to the subject an effective amount of anantibody or antibody fragment that binds a fragment set forth in SEQ IDNO: 45 of apoB100, and the antibody contains one or more of HCDR1,HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID Nos.:318-323, respectively.

One aspect of the disclosed methods provides administering an antibodycomprising at least one CDR that has the amino acid sequence of thecorresponding CDR of orticumab. More preferably, the antibody has two orthree or four or five CDRs that have the sequence of the correspondingCDRs of orticumab. If the antibody has three or four CDRs that have thesequence of the corresponding CDRs of orticumab, it is preferred if theantibody has all three heavy chain or all three light chain CDRs thathave the sequence of the corresponding CDRs of orticumab. Thus thisaspect of the methods includes an antibody comprising three light chainCDRs that have the sequence of the corresponding three light chain CDRsof orticumab, or three heavy chain CDRs that have the sequence of thecorresponding three heavy chain CDRs of orticumab. Yet more preferably,the antibody comprises three light chain CDRs and three heavy chain CDRsthat have the sequence of the corresponding CDRs of orticumab.

If the antibody does not comprise all six CDRs that have the sequence ofthe corresponding CDRs of orticumab, it is preferred if some or all ofthe 1, 2, 3, 4 or 5 “non-identical” CDRs comprise a variant of thesequence of the corresponding CDRs of orticumab. By “a variant,” weinclude the meaning that the variant has at least 50% sequence identitywith the sequence of the corresponding CDR, more preferably at least70%, yet more preferably at least 80% or at least 90% or at least 95%.Most preferably, the variant has 96% or 97% or 98% or 99% sequenceidentity with the sequence of the corresponding CDR of orticumab.Typically the “variant” CDR sequence has 5 or 4 or 3 or 2 or only 1amino acid residue difference from the sequence of the corresponding CDRof orticumab. This aspect of the invention includes administeringorticumab for the intended treatment or prophylaxis.

The antibody containing “one or more of HCDR1, HCDR2, HCDR3, LCDR1,LCDR2 and LCDR3” encompasses embodiments that the antibody contains one,any two, any three, any four, any five or all six of the CDRs (i.e.,HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3). For example, one aspect ofthe embodiment provides that the administered antibody contains HCDR1 asset forth in SEQ ID NO: 318. Another aspect provides that theadministered antibody contains HCDR2 as set forth in SEQ ID NO: 319.Another aspect provides that the administered antibody contains HCDR3 asset forth in SEQ ID NO: 320. Yet another aspect provides that theadministered antibody contains LCDR1 as set forth in SEQ ID NO: 321.Another aspect provides that the administered antibody contains LCDR2 asset forth in SEQ ID NO: 322. Another aspect provides that theadministered antibody contains LCDR3 as set forth in SEQ ID NO:323. Yetanother aspect provides that the administered antibody contains HCDR1 asset forth in SEQ ID NO:318 and HCDR2 as set forth in SEQ ID NO: 319.Another aspect provides that the administered antibody contains HCDR1 asset forth in SEQ ID NO:318 and HCDR3 as set forth in SEQ ID NO: 320.Another aspect provides that the administered antibody contains HCDR1 asset forth in SEQ ID NO:318 and LCDR1 as set forth in SEQ ID NO: 321.Another aspect provides that the administered antibody contains HCDR1 asset forth in SEQ ID NO:318 and LCDR2 as set forth in SEQ ID NO: 322.Another aspect provides that the administered antibody contains HCDR1 asset forth in SEQ ID NO:318 and LCDR3 as set forth in SEQ ID NO: 323.Another aspect provides that the administered antibody contains HCDR2 asset forth in SEQ ID NO:319 and HCDR3 as set forth in SEQ ID NO: 320.Another aspect provides that the administered antibody contains HCDR2 asset forth in SEQ ID NO:319 and LCDR1 as set forth in SEQ ID NO: 321.Another aspect provides that the administered antibody contains HCDR2 asset forth in SEQ ID NO:319 and LCDR2 as set forth in SEQ ID NO: 322.Another aspect provides that the administered antibody contains HCDR2 asset forth in SEQ ID NO:319 and LCDR3 as set forth in SEQ ID NO: 323.Another aspect provides that the administered antibody contains HCDR3 asset forth in SEQ ID NO:320 and LCDR1 as set forth in SEQ ID NO: 321.Another aspect provides that the administered antibody contains HCDR3 asset forth in SEQ ID NO:320 and LCDR2 as set forth in SEQ ID NO: 322.Another aspect provides that the administered antibody contains HCDR3 asset forth in SEQ ID NO:320 and LCDR3 as set forth in SEQ ID NO: 323.Another aspect provides that the administered antibody contains LCDR1 asset forth in SEQ ID NO:321 and LCDR2 as set forth in SEQ ID NO: 322.Another aspect provides that the administered antibody contains LCDR1 asset forth in SEQ ID NO:321 and LCDR3 as set forth in SEQ ID NO: 323.Another aspect provides that the administered antibody contains LCDR2 asset forth in SEQ ID NO:322 and LCDR3 as set forth in SEQ ID NO: 323.Another aspect provides that the administered antibody contains HCDR1,HCDR2 and HCDR3 as set forth in SEQ ID Nos.: 318-320, respectively.Another aspect provides that the administered antibody contains HCDR1,HCDR2 and LCDR1 as set forth in SEQ ID Nos.: 318, 319 and 321,respectively. Another aspect provides that the administered antibodycontains HCDR1, HCDR2 and LCDR2 as set forth in SEQ ID Nos.: 318, 319and 322, respectively. Another aspect provides that the administeredantibody contains HCDR1, HCDR2 and LCDR3 as set forth in SEQ ID Nos.:318, 319 and 323, respectively. Another aspect provides that theadministered antibody contains HCDR1, HCDR3 and LCDR1 as set forth inSEQ ID Nos.: 318, 320 and 321, respectively. Another aspect providesthat the administered antibody contains HCDR1, HCDR3 and LCDR2 as setforth in SEQ ID Nos.: 318, 320 and 322, respectively. Another aspectprovides that the administered antibody contains HCDR1, HCDR3 and LCDR3as set forth in SEQ ID Nos.: 318, 320 and 323, respectively. Anotheraspect provides that the administered antibody contains HCDR1, LCDR1 andLCDR2 as set forth in SEQ ID Nos.: 318, 322 and 323, respectively.Another aspect provides that the administered antibody contains HCDR1,LCDR1 and LCDR3 as set forth in SEQ ID Nos.: 318, 321 and 323,respectively. Another aspect provides that the administered antibodycontains HCDR1, LCDR2 and LCDR3 as set forth in SEQ ID Nos.: 318, 322and 323, respectively. Another aspect provides that the administeredantibody contains HCDR2, HCDR3 and LCDR1 as set forth in SEQ ID Nos.:319, 320 and 321, respectively. Another aspect provides that theadministered antibody contains HCDR2, HCDR3 and LCDR2 as set forth inSEQ ID Nos.: 319, 320 and 322, respectively. Another aspect providesthat the administered antibody contains HCDR2, HCDR3 and LCDR3 as setforth in SEQ ID Nos.: 319, 320 and 323, respectively. Another aspectprovides that the administered antibody contains HCDR2, LCDR1 and LCDR2as set forth in SEQ ID Nos.: 319, 321 and 322, respectively. Anotheraspect provides that the administered antibody contains HCDR2, LCDR1 andLCDR3 as set forth in SEQ ID Nos.: 319, 321 and 323, respectively.Another aspect provides that the administered antibody contains HCDR2,LCDR2 and LCDR3 as set forth in SEQ ID Nos.: 319, 322 and 323,respectively. Another aspect provides that the administered antibodycontains HCDR3, LCDR1 and LCDR2 as set forth in SEQ ID Nos.: 320, 321and 322, respectively. Another aspect provides that the administeredantibody contains HCDR3, LCDR1 and LCDR3 as set forth in SEQ ID Nos.:320, 321 and 323, respectively. Another aspect provides that theadministered antibody contains HCDR3, LCDR2 and LCDR3 as set forth inSEQ ID Nos.: 320, 322 and 323, respectively. Another aspect providesthat the administered antibody contains LCDR1, LCDR2 and LCDR3 as setforth in SEQ ID Nos.: 321-323, respectively. Yet another aspect providesthat the administered antibody contains HCDR1, HCDR2, HCDR3 and LCDR1 asset forth in SEQ ID Nos.: 318-321, respectively. Another aspect providesthat the administered antibody contains HCDR1, HCDR2, HCDR3 and LCDR2 asset forth in SEQ ID Nos.: 318-320 and 322, respectively. Another aspectprovides that the administered antibody contains HCDR1, HCDR2, HCDR3 andLCDR3 as set forth in SEQ ID Nos.: 318-320 and 323, respectively.Another aspect provides that the administered antibody contains HCDR1,HCDR2, LCDR1 and LCDR2 as set forth in SEQ ID Nos.: 318, 319, 321 and322, respectively. Another aspect provides that the administeredantibody contains HCDR1, HCDR2, LCDR1 and LCDR3 as set forth in SEQ IDNos.: 318, 319, 321 and 323, respectively. Another aspect provides thatthe administered antibody contains HCDR1, HCDR2, LCDR2 and LCDR3 as setforth in SEQ ID Nos.: 318, 319, 322 and 323, respectively. Anotheraspect provides that the administered antibody contains HCDR1, HCDR3,LCDR1 and LCDR2 as set forth in SEQ ID Nos.: 318, 320, 321 and 322,respectively. Another aspect provides that the administered antibodycontains HCDR1, HCDR3, LCDR1 and LCDR3 as set forth in SEQ ID Nos.: 318,320, 321 and 323, respectively. Another aspect provides that theadministered antibody contains HCDR1, HCDR3, LCDR2 and LCDR3 as setforth in SEQ ID Nos.: 318, 320, 322 and 323, respectively. Anotheraspect provides that the administered antibody contains HCDR1, LCDR1,LCDR2 and LCDR3 as set forth in SEQ ID Nos.: 318, 321, 322 and 323,respectively. Another aspect provides that the administered antibodycontains HCDR2, HCDR3, LCDR1 and LCDR2 as set forth in SEQ ID Nos.:319-322, respectively. Another aspect provides that the administeredantibody contains HCDR2, HCDR3, LCDR1 and LCDR3 as set forth in SEQ IDNos.: 319-321 and 323, respectively. Another aspect provides that theadministered antibody contains HCDR2, HCDR3, LCDR2 and LCDR3 as setforth in SEQ ID Nos.: 319, 320, 322 and 323, respectively. Anotheraspect provides that the administered antibody contains HCDR2, LCDR1,LCDR2 and LCDR3 as set forth in SEQ ID Nos.: 319, 321, 322 and 323,respectively. Another aspect provides that the administered antibodycontains HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID Nos.:320-323, respectively. Yet another aspect provides that the administeredantibody contains HCDR1, HCDR2, HCDR3, LCDR1 and LCDR2 as set forth inSEQ ID Nos.: 318-322 respectively. Another aspect provides that theadministered antibody contains HCDR1, HCDR2, HCDR3, LCDR1 and LCDR3 asset forth in SEQ ID Nos.: 318-321 and 323 respectively. Another aspectprovides that the administered antibody contains HCDR1, HCDR2, HCDR3,LCDR2 and LCDR3 as set forth in SEQ ID Nos.: 318-320, 322 and 323respectively. Another aspect provides that the administered antibodycontains HCDR1, HCDR2, LCDR1, LCDR2 and LCDR3 as set forth in SEQ IDNos.: 318, 319, 321-323, respectively. Another aspect provides that theadministered antibody contains HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 asset forth in SEQ ID Nos.: 318, 320-323, respectively. Another aspectprovides that the administered antibody contains HCDR2, HCDR3, LCDR1,LCDR2 and LCDR3 as set forth in SEQ ID Nos.: 319-323, respectively. Yetanother aspect provides that the administered antibody contains HCDR1,HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID Nos.:318-323, respectively.

Methods are provided of treating or reducing the severity or likelihoodof SLE in a subject, or of atherosclerosis in a subject with SLE,including administering to the subject an effective amount of anantibody or antibody fragment that binds a fragment set forth in SEQ IDNO: 45 of apoB100, and the antibody contains a variable heavy region(V_(H)) as set forth in SEQ ID NO: 324 and a variable light region(V_(L)) containing LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID Nos.:321-323, respectively. A further aspect provides that the method oftreating or reducing the severity of SLE in a subject, or ofatherosclerosis in a subject with SLE, and/or provide passive immunity,includes administering to the subject an effective amount of an antibodyor antibody fragment that binds a fragment set forth in SEQ ID NO: 45 ofapoB100, and the antibody contains a variable light region (V_(L)) ofSEQ ID NO: 325 and a variable heavy region (V_(H)) that contains HCDR1,HCDR2 and HCDR3 as set forth in SEQ ID Nos.: 318-320, respectively. Yetanother aspect of the invention provides that the method of treating,reducing the severity or likelihood of SLE in a subject, or ofatherosclerosis in a subject with SLE, includes administering to thesubject an effective amount of an antibody or antibody fragment thatbinds a fragment set forth in SEQ ID NO: 45 of apoB100, and the antibodycontains a variable heavy region (V_(H)) of SEQ ID NO: 324 and avariable light region (V_(L)) of SEQ ID NO:325.

Methods are also provided of treating or reducing the severity orlikelihood of SLE in a subject, or of atherosclerosis in a subject withSLE, which includes administering to the subject an effective amount ofan antibody or antibody fragment that binds a fragment set forth in SEQID NO: 45 of apoB100, and the antibody contains a heavy chain of SEQ IDNO: 316 and a light chain containing LCDR1, LCDR2 and LCDR3 as set forthin SEQ ID Nos.: 321-323, respectively. A further aspect of theembodiment provides that the method includes administering to thesubject an effective amount of an antibody or antibody fragment thatbinds a fragment set forth in SEQ ID NO: 45 of apoB100, and the antibodycontains a heavy chain of SEQ ID NO: 316 and a light chain that containsa variable light region (V_(L)) of SEQ ID NO: 325. Another aspect of theinvention provides the method includes administering to the subject aneffective amount of an antibody or antibody fragment that binds afragment set forth in SEQ ID NO: 45 of apoB100, and the antibodycontains a light chain of SEQ ID NO: 317 and a heavy chain that containsHCDR1, HCDR2 and HCDR3 as set forth in SEQ ID Nos.: 318-320,respectively. Yet another aspect provides the method includesadministering to the subject an effective amount of an antibody orantibody fragment that binds a fragment set forth in SEQ ID NO: 45 ofapoB100, and the antibody contains a light chain of SEQ ID NO: 317 and aheavy chain that contains a variable heavy region (V_(H)) of SEQ ID NO:324. Alternatively, the method includes administering to the subject aneffective amount of an antibody or antibody fragment that binds afragment set forth in SEQ ID NO: 45 of apoB100, and the antibodycontains a heavy chain of SEQ ID NO: 316 and a light chain of SEQ ID NO:317.

Further embodiments of the methods include administering an inhibitor ofnative LDL, oxidized LDL (oxLDL) or MDA-modified LDL, which are suitablefor treatment of a subject diagnosed with SLE and reducing the severityor likelihood of SLE, and optionally also reducing the severity or thelikelihood of developing atherosclerosis in a subject with SLE. In oneembodiment, the inhibitor is an anti-oxLDL antibody or anantigen-binding fragment thereof capable of binding an oxidized fragmentof apolipoprotein B100. In another embodiment, the inhibitor of oxidizedLDL is a small molecule, a polypeptide, a peptide, or a nucleic acidmolecule, which is capable of binding an oxidized fragment ofapolipoprotein B100. In other embodiments, the inhibitor of oxidized LDLis an antibody or an antigen-binding fragment capable of binding amalondialdehyde-modified LDL. In exemplary embodiments, the inhibitor ofoxidized or malondialdehyde-modified LDL is a monoclonal antibody, suchas orticumab, targeting an oxidized or MDA-modified form of a LDL.

Patient Selection

The methods disclosed herein, in some embodiments, include treating orinhibiting one or more forms of SLE, or of atherosclerosis in a subjectwith SLE, with administering an effective amount of orticumab to asubject in need thereof.

Some embodiments provided in the disclosed methods further includeselecting a subject showing symptoms of SLE or having been diagnosedwith SLE. For example, subjects with SLE can be characterized by havinga positive test for antinuclear antibody (ANA) in combination with oneor more symptoms such as rash (e.g., malar rash, discoid skin rash),arthritis, or edema in the ankles; having an abnormal sound called aheart friction rub or pleural friction rub; having an abnormally highcreatinine; photosensitivity, mucous membrane ulcers, arthritis,pleuritic or pericarditis, kidney abnormalities, brain irritation,blood-count abnormalities, immunologic disorder, and positiveantinuclear antibodies in the blood.

Also provided with the methods are embodiments where the subject doesnot have, has not been diagnosed with, or does not show symptoms ofatherosclerosis.

Combination Therapy

Further embodiments provide that the methods of treating or reducing theseverity or likelihood of SLE in a subject, or of atherosclerosis in asubject with SLE, include administering an effective amount of anantibody or antibody fragment in combination with another therapeuticagent to the subject. Exemplary therapeutic agents for use in thiscombination include an anti-malarial therapeutic (e.g.,hydroxychloroquine), glucocorticoid, mycophenolate mofetil, or adisease-modifying antirheumatic drug (DMARD; such as azathioprine,cyclophosphamide, cyclosporine, hydroxychloroquine sulfate, leflunomide,methotrexate, mycophenolate mofetil, and sulfasalazine).

The methods may also include administering an antibody or antibodyfragment that binds SEQ ID NO:45, another therapeutic agent, andcommonly used adjuvants to enhance absorption of the antibody or mixtureof antibodies.

In various embodiments, the composition to be administered in thedisclosed methods are formulated for delivery via any route ofadministration. For example, the methods include administration via anaerosol, nasal, oral, transmucosal, transdermal, parenteral or enteralroute. “Parenteral” refers to a route of administration that isgenerally associated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders. Via the parenteral route,the compositions may be in the form of solutions or suspensions forinfusion or for injection. Via the enteral route, the pharmaceuticalcompositions can be in the form of tablets, gel capsules, sugar-coatedtablets, syrups, suspensions, solutions, powders, granules, emulsions,microspheres or nanospheres or lipid vesicles or polymer vesiclesallowing controlled release. Typically, the compositions areadministered by injection.

Dosage

Typically, an effective amount of the anti-oxLDL or an anti-LDLantibody, or the antibody that binds SEQ ID NO:45, in the methoddisclosed herein, results in a plasma concentration of at least 4 μg/mL,preferably at least 12 μg/mL in the subject.

Embodiments provide the method of treating or reducing the severity ofSLE in a subject, or of atherosclerosis in a subject with SLE, in asubject includes administering to the subject an antibody or antibodyfragment disclosed above subcutaneously at about 330 mg/month for about3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or longer, and the subject isan adult human.

Other embodiments provide administering the antibody or antibodyfragment to treat SLE, or treat atherosclerosis in a subject with SLE,or provide passive immunity at least 5, 6, 7, or 8 mg orticumab/kg of apatient (e.g., 664 mg for an averaged human patient of 83 kg). Someembodiments provide administering the antibody or antibody fragment atbetween 5 mg orticumab/kg of a patient (e.g., 415 mg for an averagedhuman patient of 83 kg) and 8 mg/kg. Some embodiment providesadministering orticumab at a monthly dosing regimen at theabove-mentioned dosage.

Other embodiments provide administering the antibody or antibodyfragment to treat SLE, or treat atherosclerosis in a subject with SLE,weekly at no less than 2 mg/kg/week (166 mg for an averaged humanpatient of 83 kg); preferably, 4 mg/kg/week (332 mg for an averagedhuman patient of 83 kg). In another aspect, the composition of ananti-oxLDL antibody is administered biweekly at >2.5 mg/kg/two weeks(e.g., 208 mg for an averaged human patient of 83 kg). In yet anotheraspect, the composition of an anti-oxLDL antibody is administeredmonthly at about 6 mg/kg/month (e.g., about 498 mg for an averaged humanpatient of 83 kg). For example, the monthly dosing may be carried outfor 12 months or 3 months.

Yet other embodiments provide administering an antibody or antibodyfragment to treat SLE, or treat atherosclerosis in a subject with SLE,with at least an initial dose of 800-900 mg, 900-1000 mg, 1000-1100 mg,1100-1200 mg, 1200-1300 mg, 1300-1400 mg, 1400-1500 mg, or 1500-1600 mg.In some aspects, the effective amount in the method described hereinincludes an initial dose of orticumab of approximately 1000-1500 mg,followed by subsequent doses of the antibody at 700-900 mg administeredweekly for 2, 3, 4 or 5 weeks and/or even administered monthly for 1, 2or 3 months.

Other embodiments provide administering step-wise escalating doses of anantibody against native or oxidized LDL (e.g., binding SEQ ID NO:45). Inthis embodiment, an exemplary (starting) dose of a single-doseadministration of an antibody (e.g., orticumab) against native oroxidized LDL is between 0.005 and 0.01 mg/kg (e.g., intravenously); andother exemplary dosage levels to be administered in the single-doseadministration are between 0.01 and 0.15, between 0.15 and 0.75, between0.75 and 2.5, between 2.5 and 7.5, and between 7.5 and 30 mg/kg (e.g.,intravenously). For example, a starting dose of orticumab in asingle-dose intravenous administration is 0.007 mg/kg; and otherexemplary dosages can be 0.05, 0.25, 1.25, 5.0 or 15.0 mg/kg insubsequent single-dose intravenous administration. In anotherembodiment, a single-dose subcutaneous administration of an antibodyagainst native or oxidized LDL is between 0.5 and 5 mg/kg, and amultiple-dose subcutaneous administration is also between 0.5 and 5mg/kg. For example, an antibody against native or oxidized LDL at 1.25mg/kg is administered subcutaneously. In various embodiments, the dosageis administered within a specified hour range of the day in eachadministration, and each dose in a multiple-dose treatment (e.g., 4doses, 3 doses, 5 doses, or 6 doses) is administered at weekly intervalswith a time window of ±1 day. In another example, an antibody (such asorticumab) against native or oxidized LDL is administered at between 300mg and 450 mg (e.g., 360 mg) to a human subject, optionally followed byanother dose between 300 mg and 450 mg (e.g., 360 mg) to the humansubject where the second dose is at least 70 days (up to 91 days) apartfrom the first dose. The antibody (such as orticumab) may be formulatedat a concentration of 100-170 mg/mL (e.g., 150 mg/mL) and for use insubcutaneous administration without further dilution, or diluted to alarge volume for intravenous infusion.

Further embodiments include administering to a subject an effectiveamount of an antibody or antibody fragment that binds SEQ ID NO:45 andhaving a sequence of one or more of SEQ ID Nos: 316-325, which is in therange of about 10-50 μg/period, 50-100 μg/period, 100-150 μg/period,150-200 μg/period, 100-200 μg/period, 200-300 μg/period, 300-400μg/period, 400-500 μg/period, 500-600 μg/period, 600-700 μg/period,700-800 μg/period, 800-900 μg/period, 900-1000 μg/period, 1000-1100μg/period, 1100-1200 μg/period, 1200-1300 μg/period, 1300-1400μg/period, 1400-1500 μg/period, 1500-1600 μg/period, 1600-1700μg/period, 1700-1800 μg/period, 1800-1900 μg/period, 1900-2000μg/period, 2000-2100 μg/period, 2100-2200 μg/period, 2200-2300μg/period, 2300-2400 μg/period, 2400-2500 μg/period, 2500-2600μg/period, 2600-2700 μg/period, 2700-2800 μg/period, 2800-2900 μg/periodor 2900-3000 μg/period. A period is a day, a week, a month, or anotherlength of time. One aspect is the antibody (e.g., orticumab) isadministered at a weekly, biweekly or monthly frequency of any ofabove-mentioned dosage per period.

In some embodiments, the methods include administering an inhibitor ofoxidized LDL (e.g., orticumab) to the subject for 1-5 days, 1-5 weeks,1-5 months, or 1-5 years. For example, the antibody is administered tothe subject in 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 doses, each dose separated by at least 3 days, 5 days, oneweek, two weeks, one month, two months, or a combination thereof. Inother embodiments, the second dose is administered about 2-3 weeks, orabout 3 weeks after the first dose and the third dose is administeredabout 5-6 weeks or about 6 weeks after the first dose, etc. In anotherembodiment, the second dose is administered about 2-3 months, about 2months, about 3 months or about 4 months after the first dose and thethird dose is administered about 4-6 months, about 5-6 months, about 5months or about 6 months after the first dose.

Other embodiments provide administering step-wise escalating doses of anantibody against native or oxidized LDL to treat SLE, or treatatherosclerosis in a subject with SLE, or provide passive immunity. Inthis embodiment, an exemplary (starting) dose of a single-doseadministration of an antibody (e.g., orticumab) against native oroxidized LDL is between 0.005 and 0.01 mg/kg (e.g., intravenously); andother exemplary dosage levels to be administered in the single-doseadministration are between 0.01 and 0.15, between 0.15 and 0.75, between0.75 and 2.5, between 2.5 and 7.5, and between 7.5 and 30 mg/kg (e.g.,intravenously). For example, a starting dose of orticumab in asingle-dose intravenous administration is 0.007 mg/kg; and otherexemplary dosages can be 0.05, 0.25, 1.25, 5.0 or 15.0 mg/kg insubsequent single-dose intravenous administration. In anotherembodiment, a single-dose subcutaneous administration of an antibodyagainst native or oxidized LDL is between 0.5 and 5 mg/kg, and amultiple-dose subcutaneous administration is also between 0.5 and 5mg/kg. For example, an antibody against native or oxidized LDL at 1.25mg/kg is administered subcutaneously. In various embodiments, the dosageis administered within a specified hour range of the day in eachadministration, and each dose in a multiple-dose treatment (e.g., 4doses, 3 doses, 5 doses, or 6 doses) is administered at weekly intervalswith a time window of ±1 day. In another example, an antibody (such asorticumab) against native or oxidized LDL is administered at between 300mg and 450 mg (e.g., 360 mg) to a human subject, optionally followed byanother dose between 300 mg and 450 mg (e.g., 360 mg) to the humansubject where the second dose is at least 70 days (up to 91 days) apartfrom the first dose. The antibody (such as orticumab) may be formulatedat a concentration of 100-170 mg/mL (e.g., 150 mg/mL) and for use insubcutaneous administration without further dilution, or diluted to alarge volume for intravenous infusion. In some embodiments, thetherapeutically effective amount of an anti-LDL antibody or ananti-oxLDL antibody, or analogs, pharmaceutical equivalents or apeptidomimetics thereof, for use with the methods described herein isper dose: 1-10 μg/kg, 10-100 μg/kg, 100-500 μg/kg, 200-500 μg/kg,300-500 μg/kg, 400-500 μg/kg, 1-5 mg/kg, 5-10 mg/kg, 10-15 mg/kg, 15-20mg/kg, 20-25 mg/kg, 25-50 mg/kg, 50-75 mg/kg of the subject; where eachdose is administered daily, weekly, monthly, or at other intervals.

In some embodiments, an instruction manual for use, a vial for diluent,or both are also included in the kit, in addition to the one or theplurality of vials/doses of the antibody or antibody fragment to treatSLE or treat atherosclerosis in a subject diagnosed with SLE.

Pharmaceutical Composition or Medicaments

In various embodiments, the present invention provides a pharmaceuticalcomposition for use in the methods of treating, reducing the severity orlikelihood of SLE in a subject, or of atherosclerosis in a subject withSLE, described herein. The pharmaceutical composition includes aninhibitor of oxidized LDL, such as an anti-oxLDL antibody that binds toan epitope of SEQ ID NO:45 of ApoB100, and a pharmaceutically acceptablecarrier. “Pharmaceutically acceptable carrier” as used herein refers toa pharmaceutically acceptable material, composition, or vehicle that isinvolved in carrying or transporting a compound of interest from onetissue, organ, or portion of the body to another tissue, organ, orportion of the body. For example, the carrier may be a liquid or solidfiller, diluent, excipient, solvent, or encapsulating material, or acombination thereof. Examples of excipients include but are not limitedto starches, sugars, microcrystalline cellulose, diluents, granulatingagents, lubricants, binders, disintegrating agents, wetting agents,emulsifiers, coloring agents, release agents, coating agents, sweeteningagents, flavoring agents, perfuming agents, preservatives, antioxidants,plasticizers, gelling agents, thickeners, hardeners, setting agents,suspending agents, surfactants, humectants, carriers, stabilizers, andcombinations thereof. Generally, each component of the carrier must be“pharmaceutically acceptable” in that it must be compatible with theother ingredients of the formulation. It must also be suitable for usein contact with any tissues or organs with which it may come in contact,meaning that it must not carry a risk of toxicity, irritation, allergicresponse, immunogenicity, or any other complication that excessivelyoutweighs its therapeutic benefits.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Further embodiments provide that a composition or medicament for use intreating, reducing the severity of, or promoting prophylaxis of SLE, ortreating, reducing the severity of, or promoting prophylaxisatherosclerosis in a subject exhibiting symptoms of or having beendiagnosed with SLE, where the composition of medicament contains ananti-oxLDL antibody that binds to an epitope of SEQ ID NO:45 of ApoB100,as disclosed above, in an amount of between 300 mg and 400 mg,preferably about 330 mg, per dosage (or vial), optionally with apharmaceutically acceptable carrier, each (e.g., for a monthlysubcutaneous administration to a subject) for 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 months or longer. Other embodiments provide the compositionor medicament contains an anti-oxLDL antibody that binds to an epitopeof SEQ ID NO:45 of ApoB100, as disclosed above, in an amount of at least5, 6, 7, or 8 mg orticumab/kg of a patient in one dosage (or vial), andoptionally more dosages (or vials) of at least 2 mg/kg/week, at least2.5 mg/kg/two weeks, or at least 6 mg/kg/month, for 3, 4, 5, 6, 7, 8, 9,10, 11, 12 months or longer. Further embodiments provide the compositionor medicament contains the antibody (such as orticumab) at aconcentration of 100-170 mg/mL (e.g., 150 mg/mL) and for use insubcutaneous administration without further dilution, or diluted to alarge volume for intravenous infusion.

Prepare Antibodies in the Methods

In some embodiments, the aforementioned methods involve antibodies thatbind to a specific antigen epitope, where the antibodies contain one ormore defined sequences. For example, modern recombinant librarytechnology is used to prepare therapeutic antibodies against nativeApoB, oxidized ApoB or MDA-modified ApoB. While murine hybridomas cellsproduce large amounts of identical antibodies, these non-humanantibodies are recognized by human body as foreign, and as aconsequence, their efficacy and plasma half-lives are decreased inaddition to eliciting allergic reactions. To solve this problem, oneapproach is to make chimeric antibodies where the murine variabledomains of the antibody are transferred to human constant regionsresulting in an antibody that is mainly human. A further refinement ofthis approach is to develop humanized antibodies where the regions ofthe murine antibody that contacted the antigen, the so calledComplementarity Determining Regions (CDRs) are transferred to a humanantibody framework, resulting in a humanized antibody. Another approachis to produce completely human antibodies using recombinanttechnologies, which does not rely on immunization of animals to generatethe specific antibody. Instead recombinant libraries comprise a hugenumber of pre-made antibody variants and it is likely that a librarywill have at least one antibody specific for any antigen. A phagedisplay system may be used where antibody fragments are expressed,displayed, as fusions with phage coat proteins on the surface offilamentous phage particles, while the phage display systemsimultaneously carries the genetic information encoding the displayedmolecule. Phage displaying antibody fragments specific for a particularantigen may be selected through binding to the antigen in question.Isolated phage may then be amplified and the gene encoding the selectedantibody variable domains may optionally be transferred to otherantibody formats as e.g. full length immunoglobulin and expressed inhigh amounts using appropriate vectors and host cells well known in theart. The format of displayed antibody specificities on phage particlesmay differ. The most commonly used formats are Fab and single chain(scFv) both containing the variable antigen binding domains ofantibodies. The single chain format is composed of a variable heavydomain (V_(H)) linked to a variable light domain (V_(L)) via a flexiblelinker. Before use as analytical reagents, or therapeutic agents, thedisplayed antibody specificity is transferred to a soluble format, e.g.,Fab or scFv, and analyzed as such. In later steps the antibody fragmentidentified to have desirable characteristics may be transferred into yetother formats such as full length antibodies.

Antibody Production Using Hybridomas

The cell fusions are accomplished by standard procedures well known tothose skilled in the field of immunology. Fusion partner cell lines andmethods for fusing and selecting hybridomas and screening for mAbs arewell known in the art. See, e.g., Ausubel infra, Harlow infra, andColligan infra, the contents of which references are incorporatedentirely herein by reference.

An anti-oxidized LDL antibody can be produced in large quantities byinjecting hybridoma or transfectoma cells secreting the antibody intothe peritoneal cavity of mice and, after appropriate time, harvestingthe ascites fluid which contains a high titer of the mAb, and isolatingthe mAb therefrom. For such in vivo production of the mAb with anon-murine hybridoma (e.g., rat or human), hybridoma cells arepreferably grown in irradiated or athymic nude mice. Alternatively, theantibodies can be produced by culturing hybridoma or transfectoma cellsin vitro and isolating secreted mAb from the cell culture medium orrecombinantly, in eukaryotic or prokaryotic cells.

Recombinant Expression of Anti-Oxidized LDL Antibodies

Recombinant murine or chimeric murine-human or human-human antibodiesthat inhibit oxidized LDL can be provided according to the presentinvention using known techniques based on the teaching provided herein.See, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology,Wiley Interscience, N.Y. (1987, 1992, 1993); and Sambrook et al.Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989).

The DNA encoding an anti-oxidized LDL antibody can be genomic DNA orcDNA which encodes at least one of the heavy chain constant region (Hc),the heavy chain variable region (Hc), the light chain variable region(Lv) and the light chain constant regions (Lc). A convenient alternativeto the use of chromosomal gene fragments as the source of DNA encodingthe murine V region antigen-binding segment is the use of cDNA for theconstruction of chimeric immunoglobulin genes, e.g., as reported by Liuet al. (Proc. Natl. Acad. Sci., USA 84:3439 (1987) and J. Immunology139:3521 (1987). The use of cDNA requires that gene expression elementsappropriate for the host cell be combined with the gene in order toachieve synthesis of the desired protein. The use of cDNA sequences isadvantageous over genomic sequences (which contain introns), in thatcDNA sequences can be expressed in bacteria or other hosts which lackappropriate RNA splicing systems.

EXAMPLES

The following examples are not intended to limit the scope of the claimsto the invention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods which occur tothe skilled artisan are intended to fall within the scope of the presentinvention.

Example 1. Efficacy Study of CVX-12 in Female GLD.APOE^(−/−) Mice

The inventors tested the athero-protective effects of the prototypevaccine, CVX-210-B (CVX-12), comprised of a human ApoB100 derivedpeptide, P210, conjugated to a carrier protein (cBSA), and formulatedwith an adjuvant (Alum) in selected strains of mice.

As described herein, the ApoB peptide vaccine CVX-12 has shownatheroprotective benefits in mouse models of atherosclerosis. Wehypothesized that these benefits would extend to mouse models of lupuswith accelerated atherosclerosis (gld and gld.apoE−/− strains), and weinvestigated the impact of CVX-12 (Formulation 21 (CVX-210-B) formulatedat 1.0 mg conjugate/mL: P-C-A ratio is 1-1-6.9; 0.2 mL administered)immunization on atherosclerosis, splenocyte immune cell populations andcytokine production, and autoimmune disease phenotypes in these animals.CVX-12 immunization reduced the extent of atherosclerotic plaque inapoE−/− mice. Anti-nuclear antibody reactivity was reduced by CVX-12 ingld.apoE−/− mice maintained on normal chow diet.

The gld.apoE−/− mouse strain is an acceptable model of acceleratedatherosclerosis in the background of systemic lupus erythematosus. Thetest article used in this study was to determine its potentialatheroprotective effect in this model.

Experimental Methods

CVX-12 in concentration of either 2 mg/ml P210 conjugate (CVX-3-84) or2.1 mg/ml P210-B conjugate (CVX-3-123) were used in this study.Rosiglitazone (Ro) was used as a positive control.

Vehicle and test article were stored at 4° C. Test article was preparedby dispensing 0.5 mL of alum adjuvant into a 0.5 ml vial of P210-B witha sterile syringe. The combination was mixed by pipetting until uniform.Alum control was directly administered from the storage vial.

The rosiglitazone to be used as in-house positive control was preparedfor administration in normal chow diet. 8 mg rosiglitazone tablets werepulverized by mortar and pestle and incorporated into normal diet chow(50 mg rosiglitazone/1 kg food). Rosiglitazone diet was stored at 4° C.

Experimental Design

Three strains of female mice were evaluated in this study: ApoE^(−/−)(n=60), gld (n=60) and gld.ApoE^(−/−) (n=200). The total number ofanimals on study was 320. Gld mice are mice that have a mutation in Fasligand (FasL), a death factor that binds to its receptor, Fas, andinduces apoptosis. Gld mice experience accelerated autoimmune diseaseand may be used as a mouse species to study SLE.

Mice received either normal chow diet or a combination of normal andhigh cholesterol chow diet (0.20% cholesterol, 21% fat). In the lattergroup, 150 female mice, 60 ApoE^(−/−) and 90 gld.ApoE^(−/−), weremaintained on normal chow diet throughout the immunization period fromDay 0 (7 weeks of age) until Day 41. At Day 42, the diet was switched tohigh cholesterol chow and continued until euthanasia. In the normalchow-only group, 170 female mice, 60 gld and 90 gld.ApoE^(−/−), plus anadditional 20 gld.ApoE^(−/−) as a positive control group, received anormal chow diet for the duration of the study (Day 0 until euthanasia).

Each animal dosed with a test article received subcutaneous primaryimmunization in the dorsal interscapular area at Day 0 (7 weeks of age),followed by a booster on Day 21 and 35 (10 and 12 weeks of age).CVX-3-84 was used to dose ApoE^(−/−) (n=20), gld mice (n=20). CVX-3-123was used to dose gld.ApoE^(−/−) (n=30 on high cholesterol chow; n=30 onnormal chow diet).

20 gld.ApoE^(−/−) mice in the positive control group received an oraldose of rosiglitazone at 10 mg/kg/day in normal chow starting on Day 0(7 weeks of age) and continuing for the duration of the study period.Mice receiving Rosiglitazone were sacrificed at Day 126 (25 weeks ofage).

Gld, ApoE^(−/−), and gld.ApoE^(−/−) mice on normal diet were euthanizedon Day 126 (25 weeks of age). Gld.ApoE^(−/−) on high cholesterol chowdiet were sacrificed at either Day 126 (25 weeks of age) or Day 98 (21weeks of age). Due to severe disease observed in the gld.ApoE^(−/−)strain of mice receiving the high cholesterol diet, the duration of thein-life period for this group was reduced from 126 days (25 weeks ofage) to 98 days (21 weeks of age). As a result of the earlier endpointfor these mice, the time on the high cholesterol diet was reduced from84 days to 56 days.

All apoE^(−/−) and gld study mice (n=60 each) had a 1 week period ofacclimation before study enrollment.

Mice were enrolled into the study based on their date of birth, andage-matched mice were divided among the test article and control cohorts(i.e., PBS, Alum, and CVX-12) to supply date-matched controls at eachendpoint date. The health of all mice was monitored by veterinary staffon a daily basis.

Animals were dosed according to the schedule in Table 2:

TABLE 2 Test Article Administration Dose Admin. Mouse N Test ConjugateConjugate Aluminum Volume Sequence Sacrifice Model (320) Diet Formuation# Article (mg/mL) (mg/admin) (mg/admin) (mL) (day) (day) ApoE^(−/−) 20Normal Diet A PBS 0 0 0 0.2 0, 21, 35 126 day 0-41 Control HC Diet day42-126 ApoE^(−/−) 20 Normal Diet B Alum 0 0 1.38 0.2 0, 21, 35 126 day0-41 Control HC Diet day 42-126 ApoE^(−/−) 20 Normal Diet 21  CVX-210-B1.0 0.2 1.38 0.2 0, 21, 35 126 day 0-41 HC Diet day 42-126 Gld 20 NormalDiet A PBS 0 0 0 0.2 0, 21, 35 126 day 0-126 Control Gld 20 Normal DietB Alum 0 0 1.38 0.2 0, 21, 35 126 day 0-126 Contol Gld 20 Normal Diet21  CVX-210-B 1.0 0.2 1.38 0.2 0, 21, 35 126 day 0-126 gld.ApoE^(−/−) 30Normal Diet A PBS 0 0 0 0.2 0, 21, 35 98 or day 0-41 Control 126 ^(a) HCDiet day 42-98 gld.ApoE^(−/−) 30 Normal Diet B Alum 0 0 1.38 0.2 0, 21,35 98 or day 0-41 Control 126 ^(a) HC Diet day 42-98 gld.ApoE^(−/−) 30Normal Diet 21  CVX-210-B 1.0 0.2 1.38 0.2 0, 21, 35 98 or day 0-41 126^(a) HC Diet day 42-98 gld.ApoE^(−/−) 30 Normal Diet A PBS 0 0 0 0.2 0,21, 35 126 day 0-126 Control gld.ApoE^(−/−) 30 Normal Diet B Alum 0 01.38 0.2 0, 21, 35 126 day 0-126 Control gld.ApoE^(−/−) 30 Normal Diet21  CVX-210-B 1.0 0.2 1.38 0.2 0, 21, 35 126 day 0-126 gld.ApoE^(−/−) 20Normal Diet R Rosiglitazone* 0 0 0 * 0-126 126 day 0-126

In Table 2 above: 1) Alum (Imject Alum) adjuvant stock solution isprepared using a 50% dilution to a concentration of 6.9 mg Al/mL inorder to standardize administered dose volumes. 2) Formulation 21(CVX-210-B) formulated at 1.0 mg conjugate/mL: P-C-A (peptide,conjugate, adjuvant) ratio is 1-1-6.9. Formulation A is PBS control.Formulation B is Alum. 3) An asterisk (*) denotes: *Positive Control(Rosiglitazone) to be administered orally in normal chow at a dose of 10mg/kg/day. 4) A superscript “a” denotes: ^(a)Due to high stress levelsdemonstrated in the gld.ApoE^(−/−) strain of mice receiving the highcholesterol diet, the duration of the in-life period for this group wasreduced from 126 days (25 weeks of age) to 98 day (21 weeks of age).Similarly, the time on the high cholesterol diet was reduced from 84days to 56 days.

Cageside Observations:

All animals were observed for morbidity, mortality, injury, and theavailability of food and water at least twice daily. On occasion,veterinary consultations were conducted during the course of the study.All treatments and observations were recorded.

Detailed Clinical Observations:

Clinical observations were recorded on the day of dosing, three daysafter dosing, and weekly between doses.

Body Weights:

Body weights for all animals were measured and recorded prior torandomization and weekly during the study. Food intake was measured on aweekly basis per cage, and intake was calculated per mouse.

Physical Examinations:

A complete physical examination was conducted on all animals by a staffveterinarian pretest.

Serum Analysis:

Serum and urine was collected at day 63 of the study (16 weeks of age).Serum collected at day 63 of the study was analyzed for anti-P210 IgMand IgG antibodies. Levels of oxidized phospholipid was also measured ingld.apoE^(−/−) mice maintained on normal diet.

Postmortem Study Evaluations:

Serum and urine was collected at euthanasia. Endpoint serum was analyzedfor anti-nuclear antibody (ANA) titers, total cholesterol,triglycerides, anti-P210 IgM antibodies, and anti-P210 IgG antibodies.Endpoint serum from gld.ApoE^(−/−) animals maintained on normal diet wasalso analyzed for anti-cardiolipin antibodies. Anti-nuclear antibodytiters were tested using the NOVA-lite Hep2 ANA assay, and serumdilutions of 1:100, 1:1000, 1:10000, 1:30000, and 1:90000 were testedfor nuclear-localized antibody reactivity to human epithelial cells.Reactivity was detected using FITC-conjugated goat anti-mouse IgGantibody. ANAs were scored based on the presence or absence of antibodysignal at each titer. Total cholesterol was assessed using Cholesterol Ereagent according to the manufacturer's protocol. Triglyceride levelswere assessed with Triglyceride Reagent and with a glycerol standard.Anti-P210 antibodies were detected by ELISA using reconstituted P210peptide.

Lymph nodes and spleen were harvested and weighed. Splenocytes wereisolated and analyzed for T-regulatory cell populations byimmunofluorescence staining and flow cytometry. Splenocytes were alsocultured and T-cells activated in vitro by Mouse T-Activator CD3/CD28.Production of cytokines INF-γ, IL-6, IL-10, IL-12, and TGF-β byactivated T-cells were assessed with immunoassays.

Kidneys were fixed, sectioned, and stained with hematoxylin and eosin toassess glomerular tuft size and cell count. Extent of atherosclerosiswas quantified by oil red O stained en face prepared aorta, and oil redO stained aortic root sections.

Table 3 below defines the set of comparisons used in the statisticalanalyses on this study.

TABLE 3 Statistical Analysis Comparisons Group Comparison GroupFormulation A (PBS controls) All Formulation A groups from differentmouse models Formulation B (Alum) All Formulation B groups fromdifferent mouse models Formulation 21 (CVX-210-B All Formulation 21groups from different or CVX-12) mouse models Within each mouse modelFormulation 21 vs. Formulation B vs. Formulation A

Mortality:

2 apoE^(−/−) mice and 1 gld mouse, on PBS or Alum treatments, wereeuthanized due to hunched posture and decreased mobility. 19gld.apoE^(−/−) mice on normal diet, 37 gld.apoE^(−/−) mice on westerndiet, and 7 gld.apoE^(−/−) mice on rosiglitazone treatment wereeuthanized due to health concerns or found dead prior to their scheduledsacrifice. Unscheduled deaths were distributed among the treatmentgroups (PBS, Alum, and CVX-12), and CVX-12 did not impact the incidenceof unscheduled deaths.

Body and Organ Weights:

Body weight data are illustrated in FIG. 1. Average food consumptionmeasurements are presented in FIG. 2. There were no test article-relatedbody weight or food intake changes. All groups had fluctuations in bodyweight that are within the normal range for the species.

Anti-P210 IgM and IgG levels were assessed at the midpoint of the study(Day 63) and the endpoint (at euthanasia), and their levels in gld orapoE^(−/−) did not change as a result of Alum or CVX-12 treatment (Table4). Anti-P210 IgG levels in gld mice are intrinsically higher than IgGlevels in apoE^(−/−) mice. Overall anti-P210 IgM levels decreased overtime in the apoE^(−/−) mice between the midpoint and endpoint regardlessof the treatment group (PBS, Alum, and CVX-12). On the contrary,anti-P210 IgG levels increased over time in the gld mice between themidpoint and endpoint analyses in all three treatment groups (PBS, Alum,and CVX-12).

TABLE 4 Relative anti-P210 antibody levels in gld mice and apoE−/− mice.Values = anti-P210 OD at 405 nm ± SEM. Midpoint Endpoint IgM IgG IgM IgGapoE^(−/−) PBS  3.37 ± 0.014 0.73 ± 0.19 2.60 ± 0.19 ^(a) 0.47 ± 0.05  apoE^(−/−) 3.34 ± 0.15 1.27 ± 0.47 2.37 ± 0.19 ^(a) 0.47 ± 0.06   AlumapoE^(−/−) 3.29 ± 0.14 0.90 ± 0.24 2.38 ± 0.22 ^(a) 0.38 ± 0.04 ^(a)CVX-12 gld PBS 3.29 ± 0.14 1.28 ± 0.19 3.20 ± 0.19   2.75 ± 0.26 ^(a)gld Alum 3.78 ± 0.11 1.37 ± 0.53 3.34 ± 0.20   2.78 ± 0.27 ^(a) gldCVX-12 3.23 ± 0.15 1.46 ± 0.22 3.61 ± 0.12   3.14 ± 0.20 ^(a) ^(a) p <0.05 for midpoint vs endpoint

Anti-P210 antibodies did not increase in gld.apoE^(−/−) mice immunizedwith CVX-12 (Table 5). Instead, immunization with CVX-12 decreasedanti-P210 IgM in gld.apoE^(−/−) mice on western diet. Rosiglitazonetreatment had a similar effect on anti-P210 IgM antibodies, decreasingtheir levels in gld.apoE^(−/−) relative to PBS-treated mice on eithernormal or high cholesterol diet protocols.

TABLE 5 Relative anti-P210 antibody levels in gld.apoE−/− mice. Values =anti-P210 OD at 405 nm ± SEM. Midpoint Endpoint IgM IgG IgM IgGgld.apoE^(−/−) (ND) PBS 0.60 ± 0.03 0.57 ± 0.05 0.58 ± 0.04   0.66 ±0.06 gld.apoE^(−/−) (ND) Alum 0.59 ± 0.02 0.55 ± 0.04 0.50 ± 0.03 ^(a)0.61 ± 0.04 gld.apoE^(−/−) (ND) CVX-12 0.62 ± 0.03 0.59 ± 0.05 0.53 ±0.04   0.65 ± 0.04 gld.apoE^(−/−) (WD) PBS  0.76 ± 0.04^(b) 0.57 ± 0.060.66 ± 0.04   0.69 ± 0.04 gld.apoE^(−/−) (WD) Alum  0.70 ± 0.02^(b) 0.55± 0.05 0.61 ± 0.04 ^(a)   0.76 ± 0.05 ^(a) gld.apoE^(−/−) (WD) CVX-120.64 ± 0.05 0.54 ± 0.06 0.53 ± 0.03 ^(c) 0.69 ± 0.05 Rosiglitazone 0.64± 0.03 0.68 ± 0.07  0.42 ± 0.06 ^(a, c) 0.68 ± 0.07 ^(a) p < 0.05 formidpoint vs endpoint. ^(b)p < 0.05 for ND vs WD. ^(c) p < 0.05 fortreatment relative to PBS control.

Since peptides derived from ApoB100 are thought to be antigens forpathogenic T cells, it is thought that they can tolerize against theseresponses. As a readout, we would expect to see reduced antibodies toApoB100-related antigens. Here measurements of apoB, oxidizedphospholipid (OxPL), measure by E06 antibody, reveal a significantincrease in oxPL with CVX-12 administration in gld.apoE^(−/−) mice onnormal diet (FIG. 3), which demonstrates that the immunization had aneffect.

Anti-Nuclear Antibody Titer.

CVX-12 immunization reduced the anti-nuclear antibody titer ingld.apoE^(−/−) mice on normal diet (FIG. 4 compared to mice on westerndiet as shown in FIG. 23). This demonstrates that CVX-12 is beneficialfor lupus disease, an application of which has not previously beentested.

Anti-Cardiolipin Analysis.

Because anti-nuclear antibody titers were reduced in CVX-12 immunizedgld.apoE^(−/−) mice on normal diet, anti-cardiolipin antibody reactivitywas also assessed (FIG. 5). While not significantly reduced, we observeda trend towards decreased anti-cardiolipin antibodies in CVX-12immunized gld.apoE^(−/−) mice on normal diet. These data suggest alarger sample size would likely demonstrate a statistically significanteffect.

Postmortem Study Evaluations:

Aortic en face and root plaque measurements are illustrated in FIGS.6A-6D and FIGS. 7A-7C. Gld mice did not develop atherosclerotic lesions,and CVX-12 did not trigger spontaneous lesion formation in these mice.In apoE^(−/−) mice, en face coverage of atherosclerotic lesions wassimilar among treatment groups, but aortic root section measurementsindicated that atherosclerotic plaque depth was reduced by Alum andCVX-12 immunization in apoE^(−/−) mice. In gld.apoE^(−/−) mice, coverageof atherosclerotic lesions quantified by en face measurements of lesionarea was unaffected by CVX-12 relative to control mice. A trend towardsdecreased lesion area was observed in gld.apoE−/− mice maintained onnormal diet when treated with both Alum and CVX-12, however thesechanges were not statistically significant.

Organ Weights.

Spleen and lymph node weights are illustrated in FIG. 8. CVX-12immunization increased submandibular lymph node size in gld mice but hadno impact on lymph node size in apoE^(−/−) or gld.apoE^(−/−) mice.CVX-12 did not affect spleen size.

Kidney Analysis.

Kidney glomerular tuft area was measured in the autoimmunity-elevatedgld mice and gld.apoE^(−/−) mice (FIG. 9). Average glomerular tuft areawas unaffected by CVX-12 immunization in gld or gld.apoE^(−/−) mice fedeither a normal or Western diet.

Splenocyte Analysis.

CVX-12 immunization is hypothesized to impact atherosclerosis viamodulation of T-cell cytokine release. Thus cytokine release by culturedsplenocytes activated by CD3 and CD28 was assessed for the cytokinesIFNγ, IL-6, IL-10, IL-12 (p70), and TNFα (Tables 6-12). CVX-12immunization increased IL-6 and IL-10 cytokine release from spleenT-cells in apoE^(−/−) mice. CVX-12 immunization did not impact levels ofthe assayed cytokines in gld mice. Rosiglitazone decreased IFNγ and IL-6production by splenocytes from gld.apoE^(−/−) mice, whereas CVX-12treatment increased IFNγ release by splenocytes from gld.apoE^(−/−) fednormal diet chow. IL-10 release from gld.apoE^(−/−) splenocytes wasdecreased by CVX-12 immunization of gld.apoE^(−/−) mice on western diet.

TABLE 6 Cytokine Production from Spleen T-cells from apoE−/− mice. Allvalues are pg/mL. IFN-γ IL-6 IL-10 PBS Alum CVX-12 PBS Alum CVX-12 PBSAlum Unstimulated Avg 33.9 581.0 845.0 269.9 549.0 893.1* 34.8 43.1 SEM±19.5 ±385.6 ±23.0 ±121.8 ±124.8 ±182.2 ±12.3 ±9.5 Stimulated Avg 1254511411 12507 1689.6 3861.0* 2925.2* 1206.7 1390.2 SEM ±716.7 ±1027.1±683.9 ±295.4 ±828.8 ±350.3 ±144.3 ±314.3 IL-10 IL-12 (p70) TNFα CVX-12PBS Alum CVX-12 PBS Alum CVX-12 Unstimulated Avg 85.8* 52.2 12.0 12.38.0 23.3 7.5 SEM ±10.1 ±40.4 ±0.5 ±0.7 ±4.3 ±14.8 ±0.9 Stimulated Avg1676.3* 12.9 12.9 13.6 75.7 540.9 80.7 SEM ±171.3 ±0.4 ±0.6 ±0.7 ±4.0±265.1 ±4.8 *P < 0.05 compared to PBS.

TABLE 7 Cytokine Production (pg/mL) from Spleen T-cells from gld mice.IFN-γ IL-6 IL-10 PBS Alum CVX-12 PBS Alum CVX-12 PBS Alum UnstimulatedAvg 171.0 112.6 82.9 302.4 576.5 152.4 142.9 147.5 SEM ±55.9 ±48.8 ±48.1±99.7 ±323.4 ±43.9 ±34.8 ±65.8 Stimulated Avg 11145 12509 13780 2115.52290.4 1538.3 945.5 1132.5 SEM ±894.4 ±1119.7 ±1098.7 ±246.9 ±462.2±217.4 ±127.1 ±187.5 IL-10 IL-12 (p70) TNFα CVX-12 PBS Alum CVX-12 PBSAlum CVX-12 Unstimulated Avg 74.1 434.0 233.4 169.7 8.4 57.9 3.7 SEM±22.2 ±153.4 ±155.6 ±73.1 ±4.5 ±38.0 ±0.4 Stimulated Avg 1255.3 451.4142.7 254.2 46.7 106.9 49.9 SEM ±198.3 ±152.7 ±47.0 ±139.0 ±6.8 ±43.8±5.0

TABLE 8 IFN-γ Production from Spleen T-cells from gld.apoE−/− mice. Allvalues are pg/mL. Normal Diet Western Diet PBS Alum CVX-12 PBS AlumCVX-12 Rosi Unstimulated Avg 65.9 79.8 85.4 127.9 139.0 132.3 24.2* SEM±9.4 ±23.7 ±19.3 ±83.3 ±52.8 ±56.3 ±8.4 Stimulated Avg 12053.5 11111.914852.3* 12957.2 13721.5 9755.3 11419.9 SEM ±1047.4 ±1202.1 ±421.0±1431.8 ±679.1 ±1745.3 ±2002.3 *P < 0.05 compared to normal diet PBS

TABLE 9 IL-6 Production from Spleen T-cells from gld.apoE−/− mice. Allvalues are pg/mL. Normal Diet Western Diet PBS Alum CVX-12 PBS AlumCVX-12 Rosi Unstimulated Avg 612.3 542.1 674.8 572.0 1024.0 1855.2255.9* SEM ±98.8 ±121.1 ±268.3 ±127.2 ±315.5 ±731.9 ±84.6 Stimulated Avg2642.1 2972.8 2703.0 2556.8 3208.1 3476.3 385.0* SEM ±309.7 ±701.4±539.4 ±450.4 ±511.1 ±771.7 ±79.0 *P < 0.05 compared to normal diet PBS

TABLE 10 IL-10 Production from Spleen T-cells from gld.apoE−/− mice. Allvalues are pg/mL. Normal Diet Western Diet PBS Alum CVX-12 PBS AlumCVX-12 Rosi Unstimulated Avg 147.5 177.3 158.1 302.8 230.4 215.6 196.0SEM ±24.4 ±42.4 ±42.4 ±156.8 ±84.5 ±89.2 ±100.6 Stimulated Avg 1603.71472.1 1723.3 1696.9 1671.0 1023.5* 1528.3 SEM ±240.7 ±224.3 ±194.2±323.9 ±237.0 ±188.0 ±233.9 *P < 0.05 compared to WD alum and ND CVX-12

TABLE 11 IL-12 (p70) Production from Spleen T-cells from gld.apoE−/−mice. All values are pg/mL. Normal Diet Western Diet PBS Alum CVX-12 PBSAlum CVX-12 Rosi Unstimulated Avg 121.1 160.2 145.9 427.4 212.0 404.789.2 SEM ±39.7 ±51.0 ±61.8 ±281.2 ±83.2 ±223.7 ±40.3 Stimulated Avg147.9 235.0 70.1 743.1 148.7 225.3 44.7 SEM ±64.7 ±124.0 ±19.1 ±414.1±48.5 ±131.4 ±30.0

TABLE 12 TNFα Production from Spleen T-cells from gld.apoE−/− mice. Allvalues are pg/mL Normal Diet Western Diet CVX- CVX- PBS Alum 12 PBS Alum12 Rosi Unstim- Avg 9.4 29.4 11.4 7.5 25.0 4.6 8.9 ulated SEM ±1.2 ±17.0±2.1 ±1.3 ±18.6 ±0.6 ±1.5 Stim- Avg 50.1 42.9 58.3 59.0 68.4 53.5 39.5ulated SEM ±6.0 ±4.3 ±6.8 ±7.4 ±26.5 ±18.0 ±8.7

Serum Analysis:

Cholesterol and triglyceride results are displayed in FIG. 22. CVX-12and alum treatments significantly increased cholesterol levels ingld.apoE−/− mice on western diet, but did not affect cholesterol levelsin apoE−/− or gld mice nor in gld.apoE−/− mice on normal diet.Triglyceride levels were increased in gld.apoE−/− as a result ofrosiglitazone treatment, but the test article treatment did notsignificantly alter triglyceride levels relative to controls in any ofthe cohorts.

Analysis of Regulatory T-Cells:

T-regulatory cell populations within the spleen were assessed as apotential mechanism for conveying the atherosclerosis benefits of CVX-12immunization, and thus all study mice were assessed for populations ofT-regulatory CD3+CD4+CD25+FOXP3+ splenocytes and for CD8+ or CD4+splenocytes (FIG. 24). CD3+CD4+CD25+FOXP3+T-regulatory cell populationswere not significantly impacted by CVX-12 immunization in gld, apoE−/−,or gld.apoE−/− mice. gld mice produce significantly smaller CD4+ andCD8+ populations compared to apoE−/−, but no significant differencesamong the treatment groups (PBS, Alum, and CVX-12) were observed in CD4+or CD8+ cell populations in gld, apoE−/−, or gld.apoE−/− mice.

CVX-12 immunization reduced the anti-nuclear antibody titer levels ingld.apoE^(−/−) mice on normal diet, demonstrating its applicability fortreatment for patients with SLE, independent of cardiovascular diseaseinvolvement. Anti-P210 IgM was decreased by CVX-12 in gld.apoE^(−/−) onwestern diet. Glomerular tuft area, as a first measurement of kidneydysfunction, was not affected in a positive nor negative manner by Alumor CVX-12 treatment. Taken together, CVX-12 immunization in a mousemodel of lupus with lupus-associated accelerated atherosclerosis appearsto be safe, and effectively diminishes anti-nuclear antibody titer.

Example 2. Increased Levels of Soluble Death Receptors are Associatedwith Cardiovascular Disease in Systemic Lupus Erythematosus

Since dysregulation of apoptosis has been implicated in atherosclerosisthe present study investigated if plasma levels of the death receptorsFas, TRAIL receptor 2 (TRAIL-R2) and TNF-R1 are related to the presenceof cardiovascular disease (CVD) in patients with SLE.

Experimental Methods

Patients and Controls.

Patients and controls were included between January 2004 and October2013. All patients who fulfilled four or more of the 1982 revisedAmerican College of Rheumatology (ACR) classification criteria for SLE[Tan E M et al. The 1982 revised criteria for the classification ofsystemic lupus erythematosus. Arthritis Rheum 1982; 25:1271-7]. Patientswere required to be older than 18 years, otherwise there were noexclusion criteria. Population controls, individually matched to thefirst 322 SLE patients were identified in the population registry.Matching was performed within one year of age, for sex, and region ofliving. Controls were contacted and asked to participate through aletter. The only exclusion criterion among controls was a diagnosis ofSLE. Due to limitation of the analytical platform 69 randomly selectedcontrols were excluded from the present study.

Data Collection.

All patients and controls were investigated in person by arheumatologist. Traditional risk factors for CVD were tabulated.Hypertension was defined as a systolic BP>140 mm Hg and/or a diastolicBP>90 mm Hg or use of antihypertensive treatment. Diabetes wasconsidered present if patients were previously diagnosed with diabetes.History of vascular events, defined as a history of objectively verifiedmyocardial infarction, ischemic cerebrovascular disease or peripheralvascular disease was obtained though interview and review of medicalfiles. In SLE patients, age at diagnosis, duration of disease, and lupusmanifestations including autoantibodies were recorded. Lupus nephritiswas defined according to the 1982 revised ACR classification criteriafor nephritis [Tan E M, Cohen A S, Fries J F, et al. The 1982 revisedcriteria for the classification of systemic lupus erythematosus.Arthritis Rheum 1982; 25: 1271-7]. Organ damage was assessed withSystemic Lupus International Collaborating Clinics/ACR Damage index(SDI) [Gladman D, et al. The development and initial validation of theSystemic Lupus International Collaborating Clinics/American College ofRheumatology damage index for systemic lupus erythematosus. ArthritisRheum 1996; 39: 363-9]. All blood samples were taken after overnightfasting and laboratory examinations were performed blinded, either onfresh blood samples or after storage in −70° C.

Analysis of Circulating Death Receptors in Plasma.

Plasma levels of Fas, TNF-R1 and TRAIL-R2 were analyzed by the ProximityExtension Assay (PEA) technique using the Proseek Multiplex CVD^(96×96)reagents kit. Oligonucleotide-labeled antibody probe pairs were allowedto bind to their respective targets present in the plasma sample andaddition of a DNA polymerase led to an extension and joining of the twooligonucleotides and formation of a PCR template. Universal primers wereused to pre-amplify the DNA templates in parallel. Finally, theindividual DNA sequences were detected and quantified using specificprimers by microfluidic real-time quantitative PCR chip. The meancoefficients of variance for intra-assay variation and inter-assayvariation were 8% and 12% for Fas, 7% and 11% for TNF-R1, and 7% and 15%for TRAIL-R2. All samples were analyzed in the same run. Data analysiswas performed by a preprocessing normalization procedure using OlinkWizard for GenEx. All data are presented as arbitrary units (AU).General calibrator curves to calculate the approximate concentrationsare available on the OLINK homepage.

Determination of P45, P210 and 132GPI Autoantibodies.

Peptides corresponding to the amino acids from 661 to 680 (P45;IEIGLEGKGFEPTLEALFGK) and amino acids 3136-3155 (P210;KTTKQSFDLSVKAQYKKNKH) of human ApoB100 were synthesized and used inELISA. The peptides were modified by 0.5 M MDA for 3 h at 37° C. anddialyzed against PBS containing 1 mM EDTA as described [Fredrikson G N,et al. Identification of immune responses against aldehyde-modifiedpeptide sequences in Apo B-100 associated with cardiovascular disease.Arterioscler Thromb Vasc Biol 2003; 23: 872-8]. Native and MDA-modifiedpeptides diluted in PBS pH 7.4 (20 μg/ml) were absorbed to microtiterplate wells in an overnight incubation at 4° C. After washing with PBScontaining 0.05% Tween-20 (PBS-T) the coated plates were blocked withSuperBlock in TBS for 30 min at RT followed by an incubation of testplasma, diluted 1/100 in TBS-0.1% Tween-20 and 10% Superblock (TBS-T)for 2 hr at RT and overnight at 4° C. After rinsing, deposition ofautoantibodies directed to the peptide was detected using biotinylatedrabbit anti-human IgM or IgG antibodies appropriately diluted in TBS-T.After another incubation for 2 hr at RT the plates were washed and thebound biotinylated antibodies detected by alkaline phosphataseconjugated streptavidin, incubated for 2 hr at RT. The colour reactionwas developed by using phosphatase substrate kit and the absorbance at405 nm was measured after 90 and 120 min of incubation at RT for IgM andIgG, respectively. Data regarding the specificity and variability of theantibody ELISA have been published previously [Fredrikson G N, et al.Identification of immune responses against aldehyde-modified peptidesequences in apo B-100 associated with cardiovascular disease.Arterioscler Thromb Vasc Biol 2003; 23: 872-8; Fredrikson G N, et al.Autoantibody against the amino acid sequence 661-680 in apo B-100 isassociated with decreased carotid stenosis and cardiovascular events.Atherosclerosis 2007; 194: e188-92].

Anti-β₂GPI antibodies IgM/IgG were determined with the multipleximmunoassays, BioPlex 2200 APLS (Bio-Rad Laboratories Inc., Hercules,Calif., USA). Results were reported in the ranges between 1.9-160 U/mLfor IgM and 1.9-160 U/mL for IgG. Results were handled as continuousvariables. The multiplex assays are regarded as positive if ≥20 U/mL.This cut-off level corresponded to at least the 99th percentile ofhealthy blood donors.

Human Peripheral Blood Mononuclear Cell (PBMCs) Isolation.

PBMCs from SLE patients (n=6, x female and x male, age x+/−x years) andmatched healthy controls (n=6, x female and x male, age x+/−x years) aswell as from a healthy donor were isolated using FicollPaque Plus (GEhealthcare, Waukesha, Wis., USA) density gradient centrifugationaccording to manufacturer's instructions. The cells from SLE patientsand controls were resuspended in 40% autologous serum, 40% RPMI 1640medium (Thermo Fisher Scientific, Waltham, Mass., USA) and 20% dimethylsulfoxide (Sigma Aldrich, St. Louis, Mich., USA) and frozen in liquidnitrogen.

Death Receptor Activation of Human Donor PBMC.

Cells from healthy donors were seeded freshly at a density of 0.5×10⁶cells per well in complete RPMI (10 U/ml Penicillin/streptomycin, 1%L-glutamine, 1% sodium pyruvate, 1% Hepes and 0.1% mercaptoethanol) with2% FBS (HyClone South Logan, Utah, USA). For death receptor activation,IL-1β, soluble Fas ligand, TNF (Peprotech, Rocky Hill, N.J., USA) andLPS (Sigma Aldrich) were used at a concentration of 10 ng/ml, 0.5 μg/ml,10 ng/ml and 10 μg/ml respectively.

Flow Cytometry.

Cells from a healthy donor were seeded freshly at a density of 0.5×10⁶cells per well in complete RPMI (10 U/ml Penicillin/streptomycin, 1%L-glutamine, 1% sodium pyruvate, 1% Hepes and 0.1% mercaptoethanol) with2% FBS (HyClone South Logan, Utah, USA). For death receptor activation,IL-1β, soluble Fas ligand, TNFα (Peprotech, Rocky Hill, N.J., USA) andLPS (Sigma Aldrich) were used at a concentration of 10 ng/ml, 0.5 μg/ml,10 ng/ml and 10 μg/ml respectively.

Cell Supernatant Soluble Death Receptor Analysis.

Cell supernatants from death receptor activated PBMCs from a healthydonor and SLE patients/controls were used for measurement of solubleFas, TNF-R1 and TRAIL-R2 with a magnetic bead system multiplex assay(R&D Systems, Minneapolis, Minn., USA) obtained by Luminex machine(Bio-Rad). For SLE patients and controls, TNF-R1 concentration wasmeasured with ELISA (Abcam) according to manufacturer's recommendations.

Statistics.

Clinical characteristics are presented as median (interquartile range,IQR) for continuous variables and as percentages for categoricalvariables. Continuous variables that were not normally distributed werelog transformed. If not become normally distributed after logtransformation, non-parametric tests were used. Depending on data type,Students' t-test, Mann Whitney or Chi square test were used to comparedifferences between groups. Correlations were investigated throughcalculating the Spearman rank correlation coefficients.Multivariable-adjusted logistic regression models were performed toevaluate the associations between autoantibodies andcardiovascular/organ damage outcomes.

Plasma Levels of Soluble Death Receptors are Elevated in SLE Patientsand Associated with the Severity of Organ Damage.

We analyzed the plasma levels of soluble death receptors in a cohort of484 patients with SLE and 253 healthy controls. There was no significantage-difference between SLE patients and controls but the percent offemales was higher in the control group (Table 13). Circulating levelsof Fas, TRAIL-R2 and TNF-R1 were increased by 34.1%, 13.6% and 47.4%,respectively (p<0.001 for all) in patients with SLE as compared to thecontrols (Table 13).

TABLE 13 Circulating death receptor levels in SLE patients and controls.Distributions are given as median (interquartile range) or percentages.Differences between groups were analyzed by Mann-Whitney U test. Fas,TRAIL receptor 2 and TNF receptor 1 values are given as arbitrary units.SLE patients Controls TABLE 13 (n = 484) (n = 253) P Age (years) 46.4(33.7-57.6) 49.3 (36.0-58.8) ns Female sex (%) 86.7 93.3 0.006 Deathreceptors Fas 181 (143-234) 135 (114-166) <0.001 TRAIL receptor 2 2.5(1.9-3.5) 2.2 (1.9-2.7) <0.001 TNF receptor 1 5873 (4576-7750) 3984(3362-4689) <0.001

There were 69 CVD cases in the SLE group and 8 in the control group(Table 14).

TABLE 14 Clinical characteristic of SLE patients and controls.Distributions are given as median (interquartile range) or percentages.CVD data missing for 14 SLE patients. SLE patients Controls No CVD CVDNo CVD CVD TABLE 14 (n = 401) (n = 69) P (n = 245) (n = 8) P Age (yrs)43.9 (31.9-56.1) 56.8 (49.5-66.1) <0.001 50.0 (35.9-58.8) 54.3(50.1-61.6) ns Female sex % 85 91 ns 93 88 ns SLE characteristicsDisease duration year 6.5 (1.7-13.6) 17.2 (7.8-32.2) <0.001 NA NA SLICCdamage index (SDI) 1 (1-2) 3 (2-5) <0.001 NA NA Risk factors Smoking (%)49 71 0.002 48 50 ns Systolic BP (mm Hg) 120 (110-135) 130 (120-150)<0.001 120 (110-137) 140 (125-168) 0.02  Diastolic BP (mm Hg) 78 (70-80)80 (70-85) ns 75 (70-80) 85 (85-90) 0.003 Hypertension treatment (%)  28.9   59.4 <0.001   15.1 50 0.008 Body mass index (kg/m²) 23.8(21.4-27.1) 25.3 (21.9-29.8) 0.04 24.2 (22.0-27.5) 31.3 (27.0-35.1)0.001 Diabetes (%)   1.4  0 ns HDL (mmol/L) 1.3 (1.1-1.6) 1.2 (1.0-1.6)ns 1.5 (1.2-1.8) 1.4 (1.1-1.5) ns LDL (mmol/L) 3.0 (2.5-3.6) 2.9(2.3-3.6) ns 3.3 (2.6-4.0) 3.7 (3.0-4.4) ns Triglycerides (mmol/L) 1.0(0.6-1.4) 1.4 (1.0-1.7) <0.001 0.8 (0.6-1.1) 0.8 (0.7-1.7) ns Glucose4.8 (4.4-5.2) 5.1 (4.6-56) 0.001 4.9 (4.6-5.2) 5.3 (5.0-6.3) 0.02 High-sensitivity CRP 1.5 (0.6-5.0) 2.4 (1.0-7.0) 0.04 0.9 (0.5-2.2) 3.2(2.5-7.6) 0.006 Creatinine 67 (57-80) 76 (64-94) 0.001 65 (60-72) 70(56-89) ns

SLE patients with a prevalent CVD event were older, had a longerduration of SLE, a higher SLICC damage index and more cardiovascularrisk factors. In the SLE group those with prevalent CVD hadsignificantly higher levels of Fas, TRAIL-R2 and TNF-R1 than thosewithout CVD (Table 15). This difference remained significant forTRAIL-R2 and TNF-R1, but not for Fas, when controlling for the influenceof age and sex in logistic regression models. There were no significantdifferences between those with and without prevalent CVD in the controlgroup for Fas (144 (IQR 137-201) versus 134 AU (IQR 114-166)), TRAIL-R2(2.3 (IQR 2.1-2.9) versus 2.2 AU (IQR 1.9-2.7)) or TNF-R1 (4211 (IQR3956-4898) versus 3984 AU (IQR 3350-4689)). To determine if increasedlevels of soluble death receptors represent a general marker of organdamage we finally analyzed their relation with permanent organ damagemeasures as a SLICC score>1. A SLICC>1 was found to be associated withincreased plasma levels of Fas, TRAIL-R2 and TNF-R1 and thesedifferences remained statistically significant when controlling for ageand sex (Table 15).

TABLE 15 Circulating death receptor levels on SLE patients with andwithout nephritis and SLICC > 1. Distributions are given as median(interquartile range). TRAIL TNF TABLE 15 Fas receptor 2 receptor 1 NoCVD (n = 401) 177 (141-229) 2.4 (1.8-3.4) 5557 (4513-7564) CVD (n = 69)209 (162-272) 3.3 (2.2-4.5) 6562 (5480-9090) P 0.004 <0.001 0.001 OR(95% C.I.) 1.44 (0.87-2.38)  1.63 (1.16-2.29) 1.84 (1.12-3.01)  age andsex adjusted SLICC ≤ 1 170 (134-215) 2.3 (1.8-3.2) 5367 (4292-6985)SLICC > 1 209 (165-280) 3.0 (2.0-4.4) 6608 (5203-9027) P <0.001  ns<0.001  OR (95% C.I.) 2.34 (1.60-3.43)  1.56 (1.20-2.03) 2.53(1.72-3.72)  age and sex adjustedAssociation Between Soluble Death Receptors and Cardiovascular RiskFactors.

The plasma levels of soluble death receptors increased with age in bothSLE patients and controls (Table 16). In spite of this, there was only amodest or no association with SLE disease duration. Associations withblood pressure, BMI, LDL (only Fas), triglycerides, glucose, hsCRP andcreatinine were observed both in SLE and controls. There was an inverseassociation with HDL for TRAIL-R2 and TNF-R1 in the SLE group and forTNF receptor 1 in the control group. Smokers (current or former) hadhigher levels than those that had never smoked (Fas: 187 (IQR 152-247)versus 170 AU (IQR 134-219); p=0.001, TRAIL-R2: 2.5 (IQR 1.9-3.5) versus2.2 AU (IQR 1.8-3.0); p=0.004 or TNF-R1: 6252 (IQR 4738-8720) versus5386 AU (IQR 4375-6712); p=0.003).

TABLE 16 Association between risk factors and circulating death receptorlevels. Spearman rank correlation coefficients. SLE patients ControlsTRAIL TNF TRAIL TNF Fas receptor 2 receptor 1 Fas receptor 2 receptor 1Age (years) 0.26*** 0.21*** 0.19*** 0.31*** 0.23*** 0.16* Diseaseduration year 0.10* ns ns NA NA NA Systolic BP (mmHg) 0.26*** 0.21***0.23*** 0.36*** 0.22*** 0.16* Diastolic BP (mmHg) 0.17*** ns 0.13**0.29*** ns ns BMI (kg/m²) 0.20*** 0.13* 0.14** 0.21** 0.16** 0.22** HDL(mmol/L) ns −0.14** −0.19*** ns ns −0.14* LDL (mmol/L) 0.14** ns ns0.19** ns ns Triglycerides (mmol/L) 0.37*** 0.37*** 0.45*** 0.16* 0.19**ns Glucose 0.18*** ns 0.12*** 0.22*** 0.19** 0.22*** High-sensitivityCRP 0.18*** 0.29*** 0.43*** 0.22** 0.14* 0.14* Creatinine 0.35***0.27*** 0.43*** 0.15* 0.15* 0.20** *P < 0.05, **P < 0.01, ***P < 0.001.Association Between Soluble Death Receptors and Immune Biomarkers.

High levels of soluble Fas, TRAIL-R2 and TNF-R1 correlated with higherneutrophil and lower lymphocyte counts (Table 17). There were alsosignificant associations between some of the soluble death receptors andthe plasma levels of immune biomarkers of SLE such as C3dg, cardiolipinIgG and β2GP1 IgG. We have shown that antibodies against apolipoprotein(apo) B have a protective role in atherosclerosis and that SLE patientshave reduced levels of the apo B P210 IgG. Interestingly, we observed aninverse association between soluble death receptors and P210 IgG in thepresent study (Table 17).

TABLE 17 Association of circulating death receptor levels with bloodcells, complement and autoantibodies in SLE patients. Spearman rankcorrelation coefficients. TRAIL TNF TABLE 17 Fas receptor 2 receptor 1White blood cells Lymphocytes −0.15** −0.11* −0.15** Neutrophils 0.19**0.24*** 0.26*** Complement C1q −0.02 −0.09 −0.08 C3 −0.11* −0.06 −0.10*C3dg 0.34*** 0.09 0.33*** Autoantibodies Cardiolipin IgG ns 0.09* 0.15**Cardiolipin IgM ns ns ns β2GP1 IgG ns 0.09* 0.15** β2GP1 IgM ns ns nsApo B P210 IgG −0.21*** −0.15** −0.13** Apo B P210 IgM ns −0.11* ns *P <0.05, **P < 0.01, ***P < 0.001.Fas Ligand Activates Release of Soluble Death Receptors from PeripheralBlood Mononuclear Cells (PBMC)

The role of cell surface death receptors in apoptosis signaling is wellcharacterized but the factors that regulate the release of the solubleforms of these receptors have not been extensively studied. Hence, wecultured human PBMC in presence of different death receptor ligands andpro-inflammatory factors. Exposure of cells to Fas ligand stimulated therelease of Fas, and TRAIL-R2 into the culture medium, while exposure toIL-1β no effect and LPS only induced a small increase in the release ifTRAIL-R2 (FIGS. 10A and 10B). Incubation with TNF-α resulted in a modestinhibition of the release of TRAIL-R2, but had no effect on the releaseof Fas from PBMC. Fas ligand, IL-1β and LPS all stimulated the releaseof TNF-R1, whereas no effect was seen in response to TNF-α (FIG. 10C).These observations demonstrate that the release of Fas and TRAIL-R2 isinduced by activation of the Fas ligand/Fas pathway but not in responseto pro-inflammatory factors. In contrast, the release of TNF-R1 isstimulated both by Fas ligand and pro-inflammatory factors.

PBMC from SLE Patients and Healthy Controls Differ in Response to FasLigand Stimulation.

Stimulation of PBMCs from SLE patients with Fas ligand resulted in a 73%increase in the release of FAS and a seven-fold increase in TRAIL-R2release (FIG. 11A and FIG. 11B). A similar increase in the release ofTRAIL-R2 was seen in healthy controls exposed to Fas ligand, whereas theeffect on Fas was less pronounced and did not reach statisticalsignificance in paired t-test. Stimulation with Fas ligand did notactivate secretion of TNF-R1 (FIG. 11C) Incubation with Fas ligandincreased the frequency of apoptotic lymphocytes in PBMCs obtained fromhealthy controls but not in PBMCs from SLE patients (FIG. 11C).

The present study demonstrates that patients with SLE have increasedplasma levels of soluble Fas, TNF-R1 and TRAIL-R2 as compared to age-and sex-matched healthy controls. Moreover, patients with previousnephritis and higher organ damage scores had higher plasma levels ofFas, TNF-R1 and TRAIL-R2 than those with milder disease manifestations.

Activation of death receptors by their respective ligands (primarilyFasL, TNF-α and TRAIL) are of importance for several types ofphysiological apoptosis including deletion of activated T cells at theend of an immune response and killing of virus infected cells as well ascancer cells. However, dysfunctional activation of death receptorsignaling has also been associated with autoimmunity. Autoimmunelymphoproliferative syndrome is a rare disease caused by mutations inthe Fas gene leading to high expression of autoantibodies, recurrentautoimmune cytopenia as well as other autoimmune organ complications.Several lines of evidence have also implicated FasL/Fas signaling in thepathogenesis of SLE. In particular, altered FasL/Fas signaling isconsidered to play an important role in the disease process bycontributing to an impaired clearance of autoreactive lymphocytes andincreased exposure of hidden self-antigens. Mice with functionalmutations in the Fas (lpr mice) and FasL (gld mice) genes developSLE-like phenotypes. Moreover, T and B cells from SLE patients arecharacterized by increased cell surface expression of Fas and thisexpression has been shown to correlate with disease activity.

Death receptors can also be released by the cells in a soluble form. Thefactors that induce the release of soluble Fas and TRAIL-R2 have to ourknowledge previously not been studied in detail. In the present study wedemonstrate that the release of soluble Fas and TRAIL-R2 from humanPBMCs is enhanced by FasL, but not by IL-1β or TNF-α. This implies thatthe observed significant associations between soluble Fas and TRAIL-R2and CRP in SLE patients reflect inflammation caused by impairedregulation of apoptosis rather than a release of these death receptorsin response to inflammation. The cause of the increased levels ofsoluble TNF-R1 in SLE is more complex since the release of this receptorfrom PBMCs is stimulated by both FasL and pro-inflammatory factors, suchas IL-β and LPS. This suggests that increased levels of TNF-R1 may be amarker of both extrinsic apoptosis signaling and inflammation. We havepreviously studied sTNF-R1 in SLE from a systemic inflammatoryperspective and demonstrated that together with sTNF-R2 and TNF-α, it isa good marker of SLE disease activity and it was also associated withCVD. Interestingly, we did not observe an increased release of TNF-R1 inresponse to stimulation with TNF-α, which is in contrast to previousstudies on polymorphonuclear leukocytes and microvessel endothelialcells.

The soluble form of Fas has been shown to inhibit apoptosis by blockingthe binding of FasL to the membrane-bound form of Fas. Thus, it islikely that the increased levels of soluble Fas found in subjects withSLE may contribute to development of SLE disease characteristics in thesame way as inhibition of FasL/Fas signaling contributes to developmentof an SLE-like phenotype in experimental animals. In this context it isinteresting to note that FasL-induced apoptosis in PBMCs from controlsubjects was not associated with a significant increase in the amount ofsecreted Fas, while in PBMCs from SLE subjects stimulation with FasLstimulated the release of Fas without affecting apoptosis. One possibleexplanation to this observation could be that SLE PBMCs can blockFasL-induced apoptosis by releasing soluble Fas, which competes withmembrane-bound Fas for the binding of FasL. The lack of significanteffect of FasL on the release of Fas in the experiment comparing SLE andcontrol PBMCs in FIG. 11A could be seen as contradictory to the findingspresented in FIG. 10B that stimulation with FasL induced a significantincrease of Fas secretion in control PBMCs. However, it should be keptin mind that all cells in the latter experiment came from the sameindividual and that the increase was only about 10%.

SLE patients with clinical manifestations of CVD had higher levels ofsoluble death receptors than those without. Notably, an impairedregulation and handling of apoptosis represents an interesting commonfeature of SLE and atherosclerosis, the main cause of acutecardiovascular events. Enhanced apoptosis of smooth muscle cells inatherosclerotic plaques increases the risk for plaque rupture, which isconsidered as the main cause of acute myocardial infarction. However,there is experimental evidence that also inhibition of apoptoticsignaling as well as impaired clearance of apoptotic material contributeto atherosclerosis development. In line with this notion ApoE^(−/−) micelacking FasL or Fas both demonstrate enhanced atherosclerosisdevelopment. Also TNF-R1 deficient mice are more susceptible toatherosclerosis. TNF-α^(−/−) /ApoE^(−/−) mice are characterized byreduced formation of atherosclerotic plaques suggesting that while TNF-αsignaling through TNF-R1 is athero-protective the net effect of otherTNF receptors is pro-atherogenic.

The role of TRAIL-R2 in atherosclerosis remains relatively unknown butobservations that both TRAIL and TRAIL receptors are present inatherosclerotic plaques suggests that it may play a role in the diseaseprocess. The positive association between increased levels of solubledeath receptors and a SLICC score above one suggest that thesemechanisms also contribute to organ damage outside the cardiovascularsystem. These observations are thus in line with previous reports ofelevated soluble Fas levels in SLE patients with organ manifestations.

A striking observation in the present study was the clear associationbetween increased levels of soluble death receptors and factorsassociated with the metabolic syndrome including high BMI, glucose,triglycerides, systolic blood pressure and low HDL. The mechanismsresponsible these associations remain to be fully elucidated. In thepresent study we also identified an inverse association between solubledeath receptors and autoantibodies against the LDL antigen ApoB P210.High levels of this type of autoantibodies have been associated with alower risk of CVD and we recently reported that patients with SLE havereduced levels of ApoB autoantibodies. Moreover, recombinant IgG againstApo B have been shown to reduce atherosclerosis in experimental animalmodels. It is an interesting possibility that these autoantibodies insome way may reduce death receptor signaling and through this mechanismaffect atherosclerosis development.

The present findings demonstrate an increased release of the solubleforms of the apoptosis-signaling receptors Fas, TNF-R1 and TRAIL-R2 inSLE and that the plasma levels of these receptors are associated withthe presence of CVD as well as other types of SLE-related organcomplications. Our findings also suggest that the plasma levels ofsoluble Fas and TRAIL-R2 reflect signaling through membrane-bound deathreceptors and that increased levels of soluble Fas may inhibit apoptosisinduction in SLE through binding to FasL. Since impaired regulation ofapoptosis also has been shown to promote atherosclerosis development ingeneral we propose that increased expression of soluble death receptorsmay contribute to cardiovascular complications in SLE.

Example 3. Low Levels of Apolipoprotein B-100 Autoantibodies areAssociated with Increased Risk of Coronary Events

Previous smaller studies have indicated inverse associations betweenautoantibodies to oxidized low-density lipoprotein epitopes, andcardiovascular disease. The present study investigated associationsbetween autoantibodies against the apolipoprotein B-100 peptides P45 andP210, respectively, and risk of incident cardiovascular disease in alarge population-based cohort.

Apolipoprotein B-100 autoantibodies were analyzed by ELISA in aprospective study, including 5393 individuals (aged 46-68 years)belonging to the cardiovascular arm of the Malmö Diet and Cancer studywith a follow-up time of >15 years. Subjects that suffered an acutecoronary event during follow-up (n=382) had lower levels at baseline ofIgM autoantibodies recognizing the native and malondialdehyde-modifiedapolipoprotein B-100 peptides P45 and P210 and also lower IgG levelsrecognizing native P210, whereas no association was found with risk forstroke (n=317). Subjects in the highest compared with lowest tertile ofIgM-P45_(MDA) (hazard ratio [95% confidence interval]: 0.72 [0.55,0.94]; P=0.017) and IgG-P210_(native) (hazard ratio [95% confidenceinterval]: 0.73 [0.56, 0.97]; P=0.029) had lower risk for incidentcoronary events after adjustment for cardiovascular risk factors in Coxproportional hazard regression models. Moreover, subjects with highlevels of IgG-P210_(native) were less likely to have carotid plaques asassessed by ultrasonography at baseline (odds ratio=0.81, 95% confidenceinterval 0.70-0.95, P=0.008 after adjustment for risk factors).

This large prospective study demonstrates that subjects with high levelsof apolipoprotein B-100 autoantibodies have a lower risk of coronaryevents supporting a protective role of these autoantibodies incardiovascular disease.

We investigated the relationships of plasma levels of IgM and IgGautoantibodies against native P45 (IgMP45 native and IgG-P45native),native P210 (IgM-P210native and IgG-P210native), MDA-modified P45(IgM-P45MDA and IgGP45 MDA), and MDA-modified P210 (IgM-P210MDA andIgGP210MDA) and incidence of cardiovascular events. The findingsestablish that high levels of ApoB100 autoantibodies are associated witha lower incidence of coronary events.

Experimental Methods

The Malmö Diet and Cancer Study (MDCS) is a prospective population-basedcohort (n=28,449) study examining the association between diet andcancer [Berglund G, et al. The Malmo Diet and Cancer Study. Design andfeasibility. J Intern Med. 1993; 233:45-51]. Subjects born between 1926and 1945 and living in Malmö were eligible for inclusion in the study.Between October 1991 and February 1994, every other participant was alsoinvited to take part in a sub-study focusing on the epidemiology ofcarotid artery disease (MDCS cardiovascular cohort, n=6,103) [Hedblad B,et al. Relation between insulin resistance and carotid intima-mediathickness and stenosis in non-diabetic subjects. Results from across-sectional study in Malmo, Sweden. Diabet Med. 2000; 17:299-307].In the present study, plasma samples for assessments of ApoB100autoantibodies were available in a random subsample of 5,393 subjectsparticipating in the cardiovascular cohort of MDCS (FIG. 12), aged 46 to68 years old (mean age 57.6). Participants were followed from baselineexamination until first event of cardiovascular disease (CVD),emigration from Sweden or death, up until Dec. 31, 2009. Ascertainmentof cases and validity of the registries used (the Swedish DischargeRegistry, the Stroke Register of Malmö and the Cause of Death Registryof Sweden) have been proven to be high. A coronary event was defined asa fatal or non-fatal myocardial infarction, on the basis of theInternational Classification of Diseases 9th and 10th revisions (ICD-9and ICD-10) codes 410 and I21, respectively. Death due to ischemic heartdisease was defined on the basis of codes 412 and 414 (ICD-9) or I22,I23 and I25 (ICD-10). A stroke event was defined as a fatal- ornon-fatal stroke (ICD-9: 430, 431, 434 and 436), hemorrhagic stroke asICD-9: 430, 431 or ICD-10: I60, I61 and ischemic stroke as ICD-9:434,436 or ICD-10: I63. Throughout the follow-up period 668 incident firstevent CVD cases (398 coronary events and 329 strokes, whichever camefirst) were identified. Within the 398 incident coronary events, 66fatal myocardial infarctions, 293 non-fatal myocardial infarctions and39 ischemic heart diseases were identified. Moreover, within the 329incident stroke events, 25 fatal and 304 non-fatal strokes, whereof 269ischemic strokes, 52 hemorrhagic strokes and 8 unspecified wererecognized. Hypertension was defined as blood pressure≥140/90 mmHg orblood pressure lowering medication, high cholesterol as >5 mmol/L,smoking as current smoking. Blood pressure, smoking habits and lipidlevels were determined as previously described [Hedblad B, et al.Relation between insulin resistance and carotid intima-media thicknessand stenosis in non-diabetic subjects. Results from a cross-sectionalstudy in Malmo, Sweden. Diabet Med. 2000; 17:299-307]. The study wasapproved by the Regional Ethical Review Board in Lund (LU 51-90) and wasconducted in accordance with the Helsinki Declaration. All subjects gavewritten consent. The reporting of this cohort study is in accordancewith the STROBE guidelines.

B-mode ultrasound. Analysis of the common and bifurcation carotidarteries was performed using an Acuson 128 CT system with a 7-MHztransducer as described previously [Hedblad B, Nilsson P, Janzon L andBerglund G. Relation between insulin resistance and carotid intima-mediathickness and stenosis in non-diabetic subjects. Results from across-sectional study in Malmo, Sweden. Diabet Med. 2000; 17:299-307].Briefly, the right carotid bifurcation was scanned within a predefinedwindow comprising 3 cm of the distal common carotid artery, thebifurcation, and 1 cm of the internal and external carotid arteries. Allimages for measurement of plaque thickness were obtained in thelongitudinal projection showing the thickest intima-media complex.Plaque was defined as a focal thickening of the intima-media exceeding1.2 mm.

Determination of P45 and P210 Autoantibodies.

Peptides corresponding to the amino acids from 661 to 680 (P45;IEIGLEGKGFEPTLEALFGK, SEQ ID NO: 45) and amino acids 3136-3155 (P210;KTTKQSFDLSVKAQYKKNKH, SEQ ID NO: 210) of human ApoB100 were synthesized(K J Ross Petersen A S, Horsholm, Denmark) and used in ELISA. Thepeptides were modified by 0.5 M MDA for 3 h at 37° C. and dialyzedagainst PBS containing 1 mM EDTA as described [Palinski W, Witztum J L.Immune responses to oxidative neoepitopes on LDL and phospholipidsmodulate the development of atherosclerosis. J Intern Med. 2000;247:371-380]. Native and MDA-modified peptides diluted in PBS pH 7.4 (20μg/ml) were absorbed to microtiter plate wells (Nunc MaxiSorp, Nunc,Roskilde, Denmark) in an overnight incubation at 4° C. After washingwith PBS containing 0.05% Tween-20 (PBS-T) the coated plates wereblocked with SuperBlock in TBS (Thermo Scientific, #37535) for 30 min atroom temperature (RT) followed by an incubation of test plasma, diluted1/100 in TBS containing 0.1% Tween-20 (Thermo Scientific, #28320) and10% Superblock (TBS-T) for 2 h at RT and overnight at 4° C. Afterrinsing, deposition of autoantibodies directed to the peptide wasdetected using biotinylated rabbit polyclonal secondary antibody tohuman IgG (Abcam ab7159) or IgM antibodies (ICN 67-321, Biomedicals,Inc., Aurora, Ohio) appropriately diluted in TBST. After anotherincubation for 2 h at RT the plates were washed and the boundbiotinylated antibodies detected by alkaline phosphatase conjugatedstreptavidin (BioLegend 405211), incubated for 2 h at RT. The colourreaction was developed by using phosphatase substrate kit (ThermoScientific, #37620) and the absorbance at 405 nm was measured after 90and 120 min for IgM and IgG, respectively, of incubation at RT. A ratiobetween the absorbance unit of the individual plasma sample and theabsorbance unit of the control plasma (a plasma pool from voluntaryhealthy blood donors run on each ELISA-plate) was calculated and usedfor the analysis. Data regarding the specificity and variability of theantibody ELISA have been published previously [Fredrikson G N, et al.Identification of immune responses against aldehydemodified peptidesequences in apoB associated with cardiovascular disease. ArteriosclerThromb Vasc Biol. 2003; 23:872-878; Fredrikson G N, et al. Autoantibodyagainst the amino acid sequence 661-680 in ApoB100 is associated withdecreased carotid stenosis and cardiovascular events. Atherosclerosis.2007; 194:e188-e192]. In the present study, the intra-assay coefficientof variation was 9-10% for all eight apo-B100 autoantibodies and theinter-assay coefficient 25% for IgM-P45_(native) and IgM-P45_(MDA), 23%for IgG-P45_(native) and IgG-P45_(MDA), 13% for IgM-P210_(native) andIgM-P210_(MDA), 22% for IgG-P210_(native) and IgG-P210_(MDA),respectively. The intra-assay coefficient of variation was calculated byusing the absorbance values of the control plasma pool run on each plateof the same day and the inter-assay by using all the absorbance valuesof the control plasma pool run during the whole period of analysis.

Statistics. Clinical characteristics are presented as median(interquartile range, IQR) for continuous variables and as percentagesfor categorical variables. The 182 missing data due to technical reasonsin the measurements of ApoB100 autoantibody levels were excluded fromanalysis (FIG. 12). Skewed continuous variables were logarithmicallytransformed. An independent sample t test was used to assess normallydistributed continuous variables and a Chi-square test for proportionsbetween subjects with and without an incident cardiovascular event.Non-parametric test (Mann-Whitney) was used to assess non-normallydistributed continuous variables. Spearman's correlation coefficient wasused to examine the relationship among continuous variables asappropriate. No correction for multiple testing of the eight apo-B100autoantibodies has been performed. Several of the baseline clinicalcharacteristics that significantly differed between cases and controls(Table 18) were selected as potential confounders: age, sex,triglycerides, LDL, HDL (but not cholesterol, LDL/HDL ratio or apoB),hs-CRP, current smoking, prevalent diabetes mellitus (but not glucose orHbA1c), systolic blood pressure (but not diastolic blood pressure) andblood pressure lowering medication. The confounders not included arerepresented by related risk factors included in the model. A linearregression model was used to calculate independent associations andlogistic regression analyses (with the selected potential confoundersincluded as correction variables) were used to determine associationsbetween ApoB100 autoantibodies and presence of carotid plaques. TheKaplan-Meier method was used to evaluate rates of coronary or strokeevent-free survival of ApoB100 autoantibody tertiles, and differenceswere analyzed by log rank test. Cox proportional hazard regressionmodels were used to compare incidence of coronary or stroke eventsbetween autoantibody tertiles and to calculate risk factor adjustedhazard ratios (95% confidence interval, CI). Plots of the hazardfunction in different groups over time did not indicate that theproportional-hazards assumption was violated.

Characteristics of the Study Cohort. In this cohort of 5393 individuals(aged 46-68 years) from the cardiovascular arm of MDCS (FIG. 12), 398incident coronary events (66 fatal and 293 nonfatal myocardialinfarctions and 39 ischemic heart disease) and 329 incident strokeevents (25 fatal and 304 nonfatal strokes, whereof 269 ischemic strokes,52 hemorrhagic strokes, and 8 unspecified) were registered duringfollow-up until Dec. 31, 2009. Baseline characteristics of noncases andCVD cases are shown in Table 18. In comparison with noncases, those withincident CVD (coronary or stroke events) were older; included moremales, more smokers, and hypertensive individuals; and had a higherprevalence of diabetes mellitus. Also blood lipids, hemoglobin A1c, andhigh sensitive C-reactive protein were increased among CVD cases (Table18).

TABLE 18 Baseline Clinical Characteristics of the Study Cohort: NoncasesCases Cases (n = 4666) (n = 398),* Coronary (n = 329),* Stroke Age atscreening, y 57.6 (52.2-62.5) 61.6 (57.2-64.7)† 62.1 (58.0-64.4)† Sex, %men 39.8 61.6† 50.8† Current smoker, % 26.1 33.8† 32.8† Diabetesmellitus, %§ 7.4 19.8† 18.2† Hypertension, %∥ 62.5 81.2† 85.1† Glucose,mmol/L 4.9 (4.6-5.3) 5.1 (4.7-5.6)† 5.1 (4.7-5.5)† Triglycerides, mmol/L1.2 (0.9-1.6) 1.4 (1.0-1.9)† 1.3 (1.0-1.9)† Cholesterol, mmol/L 6.1(5.4-6.8) 6.3 (5.6-7.0)† 6.1 (5.5-6.9) HDL, mmol/L 1.4 (1.1-1.6) 1.2(1.0-1.4)† 1.2 (1.0-1.4)† LDL, mmol/L 4.1 (3.5-4.8) 4.4 (3.7-5.0)† 4.2(3.6-4.9) LDL/HDL, ratio 3.1 (2.3-3.9) 3.8 (2.9-4.7)† 3.6 (2.6-4.4)†apoB, mg/L¶ 1042 (883-1205) 1136 (997-1266)† 1074 (923-1232) SystolicBP, mm Hg 140 (126-152) 150 (138-160)† 150 (140-160)† Diastolic BP, mmHg 86 (80-92) 90 (82-95)† 90 (85-98)† HbA1c, % 4.8 (4.5-5.1) 5.0(4.6-5.4)† 5.0 (4.6-5.3)† hs-CRP, mg/L 1.3 (0.7-2.7) 2.0 (1.0-4.1)†(1.0-3.8)†

For Table 18 Above: Values presented as median and interquartile rangeor percentages. ApoB indicates apolipoprotein B; BP, blood pressure;HbA1c, Hemoglobin A1c; HDL, high-density lipoprotein; hs-CRP,high-sensitive C-reactive protein; and LDL, low-density lipoprotein.*Mann-Whitney test or χ2 test for categorical data. †P<0.001. ‡P<0.05. §History of diabetes mellitus, medication, or fasting glucose≥6.1 mmol/L.¶Blood pressure≥140/90 mm Hg or blood pressure-lowering treatment.noncases, 167 coronary, and 137 stroke events.

ApoB100 Autoantibodies and Incident CVD. CVD cases were found to havelower levels of IgM against both native and MDA-modified ApoB100 P45 andP210, respectively, as well as lower levels of IgG against nativeApoB100 P210, whereas the other IgG autoantibody levels did not differbetween groups. When subjects with incident coronary events and incidentstroke were analyzed separately, the same pattern of differences wasobserved between noncases and those with coronary events. However, noassociation was seen between ApoB100 peptide autoantibodies and risk fortotal stroke (Table 19).

TABLE 19 Ratio of ApoB100 Autoantibody Levels in Noncases and CVD Cases:Noncases Cases Cases ApoB100 Abs, (n = 4512) (n = 382), Coronary (n =317), Stroke IgM-P45_(native) 0.307 (0.135-0.579) 0.237 (0.089-0.497)†0.292 (0.119-0.512) IgM-P45_(MDA) 0.434 (0.260-0.685) 0.360(0.211-0.588)† 0.406 (0.243-0.605) IgM-P210_(native) 0.668 (0.518-0.818)0.649 (0.493-0.781)‡ 0.659 (0.488-0.811) IgM-P210_(MDA) 0.750(0.606-0.881) 0.721 (0.571-0.852)† 0.723 (0.593-0.853) IgG-P45_(native)0.231 (0.105-0.468) 0.217 (0.092-0.452)  0.224 (0.090-0.455)IgG-P45_(MDA) 0.368 (0.241-0.564) 0.341 (0.226-0.530)  0.351(0.228-0.575) IgG-P210_(native) 0.395 (0.290-0.530) 0.372 (0.266-0.478)†0.392 (0.275 0.520) IgG-P210_(MDA) 0.649 (0.489-0.830) 0.619(0.465-0.840)  0.651 (0.462-0.873)

For Table 19 Above: Abs indicates antibodies; ApoB100, apolipoproteinB-100; CVD, cardiovascular disease; IgG, immunoglobulin; IgM,immunoglobulin M; and MDA, malondialdehyde. *Ratio of the individualplasma sample and the control plasma pool. Values presented as medianand interquartile range of the ratio of the ApoB100 autoantibody levels.Skewed variables were log-transformed before analysis. †P<0.01, Studentt test. ‡P<0.05.

Also, no association was detected when only individuals with incidentischemic stroke were analyzed.

To determine if there were time-dependent associations betweenautoantibodies and incident coronary events, the autoantibody levelswere divided into tertiles that were plotted into Kaplan-Meier survivalcurves. The survival curves revealed a significant positive linear trendover tertiles of the IgM-P45_(native), IgM-P45_(MDA), IgM-P210_(native),IgM-P210_(MDA), and IgG-P210_(native) autoantibodies (Log rank [MantelCox] tests, P for linear trend<0.05; FIG. 13A-13E). No significantlinear trend was found for IgG-P45_(native), IgG-P45_(MDA), orIgG-P210_(MDA). In a Cox proportional hazard regression model, asignificant association was identified between high levels ofIgM-P45_(MDA) and lower risk of incident coronary events afteradjustment for several potential confounders, for example, age, sex,LDL, HDL, triglycerides, high-sensitive C-reactive protein, currentsmoking, prevalent diabetes mellitus, blood pressure-loweringmedication, and systolic blood pressure (hazard ratio [95% confidenceinterval (CI)]: 0.72 [0.55, 0.94], P=0.02 for the third tertile versusfirst; Table 20). Furthermore, a significant association between highlevels of IgG-P210_(native) and decreased risk of coronary events wasfound after adjustment of the model with the same risk factors (hazardratio [95% CI]: 0.73 [0.56, 0.97], P=0.03; for the third tertile versusfirst; Table 20).

TABLE 20 Hazard Ratios (HR) and 95% Confidence Intervals (CI) forIncident Coronary Event by Tertiles of IgM-P45_(MDA) andIgG-P210_(native) Autoantibodies: Model First Tertile Second TertileThird Tertile P for Trend IgM-P45_(MDA), ratio* <0.3130.313-0.578 >0.579 Coronary events, numbers 159 126 97 <0.0001 1 Cornonyevents, HR (95% 1.00  0.79 (0.62-0.99)† 0.59 (0.46-0.76)‡ 0.001 CI) 2Cornony events, HR (95% 1.00 0.94 (0.73-1.20) 0.72 (0.55-0.94)† 0.112CI) IgM-P210_(native), ratio* <0.324 0.324-0.475 >0.476 Coronary events,numbers 148 137 97 0.001 1 Cornony events, HR (95% 1.00 0.91 (0.72-1.15)0.66 (0.51-0.85)‡ 0.005 CI) 2 Cornony events, HR (95% 1.00 1.10(0.86-1.40) 0.73 (0.56-0.97)† 0.051 CI)

Table 20 above: Associations between tertiles of IgM-P45_(MDA) andIgG-P210_(native) autoantibodies and incident coronary event wascalculated using Cox proportional hazard regression unadjusted (Model 1)and adjusted for age, sex, LDL, HDL, systolic blood pressure,triglycerides, hs-CRP, current smoking, blood pressure-loweringmedication, and prevalent diabetes mellitus (Model 2). Significantassociations between number of events and tertiles of autoantibodieswere determined with a χ2 test for linear trend. CI indicates confidenceinterval; HbA1c, Hemoglobin A1c; HDL, high-density lipoprotein; HR,hazard ration; hs-CRP, high-sensitive C-reactive protein; IgG,immonoglobulin; IgM, immonoglobulin M; LDL, low-density lipoprotein; andMDA, malondialdehyde. *Ratio of the individual plasma sample and thecontrol plasma pool. †P<0.05. ‡P<0.001 vs first tertile, highlighted inbold.

No significant differences between tertiles for the other ApoB100autoantibodies were detected (Table 21).

TABLE 21 Hazard ratios (HR) and 95% confidence intervals (CI) forincident coronary event by tertiles of apo-B100 autoantibodies: 1sttertile 2nd tertile 3rd tertile P for trend IgM-P45_(native), ratio†Coronary events, numbers 137 109  95 0.004 Coronary events HR (95% CI)1.00 0.84 (0.64-1.09) 0.76 (0.58-1.03) 0.010 IgG-P45_(native), ratio†Coronary events, numbers 140 119 123 0.269 Coronary events HR (95% CI)1.00 0.87 (0.67-1.14) 1.02 (0.79-1.32) 0.167 IgG-P45_(MDA), ratio†Coronary events, numbers 138 118 113 0.098 Coronary events HR (95% CI)1.00 0.85 (0.66-1.11) 0.89 (0.68-1.16) 0.892 IgM-P210_(native) ratio†Coronary events, numbers 144 129 109 0.023 Coronary events HR (95% CI)1.00 1.07 (0.83-1.38) 0.90 (0.67-1.18) 0.298 IgM-P210_(MDA) ratio†Coronary events, numbers 147 121 114 0.032 Coronary events HR (95% CI)1.00 0.95 (0.73-1.23) 1.04 (0.80-1.35) 0.368 IgG-P210_(MDA) ratio†Coronary events, numbers 144 113 125 0.216 Coronary events HR (95% CI)1.00 0.81 (0.63-1.06) 0.96 (0.74-1.23) 0.289

Table 21 above: Associations between tertiles of ApoB100 autoantibodiesand incident coronary event was calculated using Cox proportional hazardregression adjusted for age, sex, LDL, HDL, systolic blood pressure,triglycerides, hs-CRP, current smoking, blood pressure loweringmedication and prevalent diabetes. Significant associations betweennumber of events and tertiles of autoantibodies were determined with aχ2 test for linear trend. Tertiles of the ratio of the individual plasmasample and the control plasma pool. Significant P-values for trendacross tertiles are highlighted in bold text.

The association between high levels of IgM-P45_(MDA) and lower risk ofincident coronary events was independent of the levels of theIgG-antibody recognizing the same antigen (IgG-P45_(MDA); hazard ratio[95% CI]: 0.59 [0.46, 0.76]; P<0.001). In line, high levels ofIgG-P210_(native) were independent of the levels of IgM-P210_(native)(hazard ratio [95% CI]: 0.71 [0.54, 0.93]; P=0.01). All together, theresults suggest that some of the ApoB100 autoantibodies are associatedwith a lower incidence of coronary events.

Individuals with presence of carotid plaques had lower levels of 3 ofthe apo-B100 autoantibodies; IgM-P210_(native) (0.66±0.21 absorbanceratio versus 0.68±0.22; P<0.01), IgM-P210_(MDA) (0.73±0.20 absorbanceratio versus 0.75±0.20; P<0.001), and IgG-P210_(native) (0.41±0.22absorbance ratio versus 0.44±0.22; P<0.001), in comparison to subjectswith no carotid plaques. Moreover, chi-squared tests identifiedsignificant linear trends for presence of carotid plaques across thetertiles of these 3 ApoB100 autoantibodies and also in IgG-P45_(MDA)(Table 22).

TABLE 22 Associations between presence of carotid plaques and tertilesof ApoB100 autoantibodies. 1st tertile 2nd tertile 3rd tertile P fortrend IgM-P45_(native), ratio† Carotid plaques n (%) 689 (45.4) 659(43.3) 654 (43.0) 0.169 IgM-P45_(MDA), ratio† Carotid plaques n (%) 768(46.3) 702 (42.2) 731 (43.5) 0.114 IgG-P45_(native), ratio† Carotidplaques n (%) 749 (44.7) 736 (44.4) 716 (43.0) 0.319 IgG-P45_(MDA),ratio† Carotid plaques n (%) 760 (46.1) 713 (43.5) 695 (42.5) 0.039IgM-P210_(native)ratio† Carotid plaques n (%) 770 (46.0) 732 (44.3) 699(41.7) 0.011 IgM-P210_(MDA) ratio† Carotid plaques n (%) 778 (46.7) 742(44.4) 681 (40.9) 0.001 IgG-P210_(native) ratio† Carotid plaques n (%)801 (48.1) 729 (43.7) 671 (40.2) <0.001 IgG-P210_(MDA) ratio† Carotidplaques n (%) 748 (45.0) 721 (43.1) 732 (43.9) 0.525

Table 22 above: Numbers represent individuals with presence of carotidplaques (n) and the percentage (%) the percent of individuals withpresence of carotid plaques within the tertile, respectively.Significant associations between presence of carotid plaques andtertiles of autoantibodies were determined with a χ² test for lineartrend. ^(†)Tertiles of the ratio of the individual plasma sample and thecontrol plasma pool. Significant P-values for trend across tertiles arehighlighted in bold text.

In a logistic regression model (adjusted for the same risk factors asmentioned above), there were fewer individuals with presence of carotidplaques in the upper compared with the lowest tertile ofIgG-P210_(native) (OR=0.813, 95% CI 0.70-0.95; P=0.008), whereas noassociations were found for the other autoantibodies. All autoantibodiesexcept for IgM-P45_(native) correlated inversely with LDL levels,whereas only antibodies recognizing P210 showed a significant inversecorrelation with apoB (Table 23).

TABLE 23 Spearman Bivariate Correlations (r) Between ApoB100Autoantibody Levels and LDL or ApoB. Apo B-100 Abs, Ratio* LDL,† mmol/LapoB,‡ mg/L IgM-P45_(native) NS NS IgM-P45_(MDA) r = −0.032§ NSIgM-P210_(native) R = −0.064 || R = −0.080 || IgM-P210_(MDA) R = −0.046R = −0.083 || IgG-P45_(native) R = −0.032§ NS IgG-P45_(MDA) R = −0.04 NSIgG-P210_(native) R = −0.069 || R = −0.083 || IgG-P210_(native) R =−0.042 R = −0.047§

For Table 23 above: Abs indicates antibodies; ApoB100, apolipoproteinB-100; IgG, immonoglobulin; IgM, immonoglobulin M; LDL, low-densitylipoprotein; MDA, malondialdehyde; and NS, not significant. *Ratio ofthe individual plasma sample and the control plasma pool. †Includes 4512noncases, 382 coronary, and 317 stroke events. ‡Includes 1842 noncases,167 coronary, and 137 stroke events. § P<0.05. ¶P<0.01. ∥P<0.001.

This Swedish prospective population-based study including 5393 whiteindividuals represents the hitherto largest study investigating the roleof autoantibodies recognizing ApoB100 peptides in CVD. Several of theprevious studies have had retrospective case-control design and includedsmall selected patient populations, such as individuals demonstratingdifferent established cardiovascular risk factors. The present studydesign, however, made it possible to determine whether the autoantibodylevels can predict risk of future cardiovascular events in subjects ofthe general community. The findings verified that IgG autoantibodiesrecognizing the native form of P210 are cross-sectionally associatedwith a lower presence of carotid plaques. Moreover, the IgM-P45_(MDA)and the IgG-P210_(native) autoantibodies, respectively, demonstratedindependent association with lower risk for a future coronary event.

Previous studies analyzing autoantibodies against the whole oxidized LDLparticle and their associations with CVD have demonstrated contradictoryfindings. The inconclusive results may depend on difficulties in thestandardization of the antigen. During the oxidation process of the LDLparticle, neoepitopes are formed and others degraded. This process maydeviate depending on the differences in the composition of the LDLparticle isolated from diverse individuals, resulting in a poorlydefined antigen. Studies using single LDL-derived antigens asphosphorylcholine or ApoB100 peptides have revealed a clearer picture.Previous studies analyzing ApoB100 peptide autoantibodies havedemonstrated an inverse association between the autoantibodies and CVD,also in diabetic and systemic lupus erythematosus patients. Thus, theclinical studies suggest a protective role of antibodies recognizingspecific antigens in oxidized LDL. This is supported by experimentalstudies, where treatment of atherosclerosis-prone mice with humanrecombinant IgG recognizing the MDA-P45 epitope was found to reduceaortic plaque area and plaque inflammation.

High plasma levels of IgG-P210_(native) have previously been associatedwith less severe coronary lesions, both in nondiabetic and diabeticpatients, lower risk of developing myocardial infarction, less severesubclinical atherosclerosis, decreased risk of postoperativecardiovascular death in carotid endarterectomy patients as well as alower prevalence of CVD in systemic lupus erythematosus patients. We nowextend these findings by demonstrating that low levels ofIgG-P210_(native) autoantibodies predict risk of coronary events in alarge population-based, prospective study. Interestingly, onlyantibodies recognizing the P210 epitope, and not the P45 epitope, showedan inverse correlation with apoB. It could be speculated that P210 maybe visible for the immune system in the intact LDL particle, whereas theP45 epitope may be hidden in the phospholipid layer and only visibleafter the oxidation process. Furthermore, it might be that theIgG-P210_(native) autoantibody recognizes a native or mildly modifiedP210 peptide and that this epitope plays a more important role inatherogenesis than the more extensively MDA-modified one.

In previous studies, we have detected an inverse association betweenhigh levels of autoantibodies recognizing P210_(MDA) and subclinicalatherosclerosis as well as less severe carotid disease in women. In bothcases, the epitope was recognized by IgM autoantibodies. An openquestion is if these represent natural IgM antibodies that havepreviously been shown to recognize other endogenously generatedstructures, such as oxidation-specific epitopes, or if they areclassical IgM antibodies. T cell-activated B2 cells are known to secreteadaptive IgMs, whereas B1 cells spontaneously secrete natural IgMantibodies. Experimental studies have suggested that B2 cells have aproatherogenic role, whereas B1 cells are atheroprotective depending ontheir secretion of natural IgM antibodies. Altogether, this may indicatethat the IgM antibodies recognizing a modified ApoB100 epitope representatheroprotective natural IgM antibodies. Interestingly, we havepreviously demonstrated that subjects with low levels of IgM recognizingmethylglyoxal-modified peptide p220 in ApoB100 have an increased risk todevelop cardiovascular events and that anti-methylglyoxal-p220 IgM isproduced by B1 cells. Another important oxidation-specific epitope onoxidized LDL, the phosphorylcholine epitope, has been found to berecognized by anti-phosphorylcholine IgM that represents an extensivelycharacterized natural IgM antibody. This natural IgM antibody againstthe phosphorylcholine epitope has been shown to confer protection inexperimental atherosclerosis in mice and also to be associated withreduced cardiovascular risk in humans. In support, the present studydemonstrated an inverse association between high levels of IgM-P45_(MDA)and reduced risk of future coronary events. High levels of this IgMantibody have also in a previous study showed an association with alower prevalence of CVD in systemic lupus erythematosus patients.Furthermore, in patients with type-2 diabetes mellitus, high levels ofIgM autoantibodies recognizing AGE-modified ApoB100 were found to beassociated with less severe coronary disease. Taken together, both IgMand T cell-dependent IgG antibodies recognizing oxidized LDL epitopesseem to have a protective role in atherogenesis, suggesting importantcontributions of both innate and adaptive immune responses. The reasonwhy only 2 of the autoantibodies recognizing ApoB100 epitopes measuredin the present study were associated with lower risk of incidentcoronary events may be that these 2 epitopes appear at different stagesof the modification of the LDL particle and become presented in a waythat is important for activation of immune responses, reflecting thatthe extent of LDL oxidation influences the plasma autoantibody levels.

Both inflammation and immune responses have been found to influence thepathogenesis of coronary artery disease, as well as the risk andcausation of stroke. Induced oxidative stress in the myocardium with asubsequent LDL oxidation may rapidly activate an antibody response inalready primed individuals and that these antibodies are consumeddirectly resulting in baseline levels the day after the event.

Levels of natural IgM antibodies to oxidation-specific epitopes haveshown inverse correlation with CVD in several human studies. Theiratheroprotective properties may be dependent on the ability to recognizethe oxidation-specific epitopes on oxidized LDL. Thus, the IgM-P45_(MDA)autoantibody may represent a natural IgM antibody explaining itsassociation with lower risk of future coronary events.

The strengths of the present study were the large size of the populationand the prospective design together with a 15-year follow-up time,including >600 events, making it unique by allowing for an evaluation ifthese ApoB100 epitope autoantibodies predict risk for futurecardiovascular events. The findings demonstrated an association betweenhigh levels of IgM-P45_(MDA) or IgG-P210_(native) autoantibodies and alower risk of coronary events. Taken together, and in the light ofprevious smaller studies, the present findings establish that highlevels of ApoB100 autoantibodies have a protective role in CVD.

Example 4. Decreased Levels of Autoantibodies Against ApolipoproteinB-100 Antigens are Associated with Cardiovascular Disease in SystemicLupus Erythematosus

Increased production of autoantibodies is a characteristic feature ofsystemic lupus erythematosus (SLE) and there is evidence that several ofthese autoantibodies may contribute to increased cardiovascular disease(CVD) in SLE. Autoantibodies against the apolipoprotein (apo) B-100peptides P45 and P210 have been associated with a lower CVD risk innon-SLE cohorts. The aim of the present study was to investigate how SLEaffects the occurrence of these potentially protective autoantibodies.The study cohort consisted of 434 SLE patients and 322 age- andsex-matched population controls. Antibodies against native andmalondialdehyde (MDA)-modified P45 and P210 were measured byenzyme-linked immunosorbant assay (ELISA). SLE patients hadsignificantly lower levels of P210 immunoglobulin (Ig)G and P45 IgM(both the native and malondialdehyde (MDA)-modified forms). SLE patientswith manifest CVD (myocardial infarction, ischaemic cerebrovasculardisease or peripheral vascular disease) had lower levels P210 IgG andP45 IgM than SLE patients without CVD. Decreased levels of theseautoantibodies were also observed in SLE patients with permanent organdamage, as assessed by the Systemic Lupus International CollaboratingClinics/American College of Rheumatology (ACR) Damage Index (SDI). Thepresent findings show that patients with SLE, a condition generallycharacterized by abundance of autoantibodies of multiple specificities,have reduced levels of antibodies against the apo B-100 antigens P45 andP210 and that the levels of these antibodies are reduced further in SLEpatients with CVD. These observations suggest the possibility that animpaired antibody-mediated removal of damaged LDL particles maycontribute to the development of vascular complications and organ damagein SLE.

In the present study we analyzed plasma levels of IgG and IgM againstnative and MDA-modified P45 and P210 in a cohort of SLE patients andmatched controls. The result demonstrates that patients with SLE havedecreased levels of P45 IgM and P210 IgG autoantibodies and that this isassociated with an increased risk of CVD and other organ complications.

Experimental Methods

Patients and Controls.

Patients and controls were included between January 2004 and October2013. All patients who fulfilled four or more of the 1982 revisedAmerican College of Rheumatology (ACR) classification criteria for SLE[Tan E M, Cohen A S, Fries J F et al. The 1982 revised criteria for theclassification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271-7] and who received care for SLE at the Department ofRheumatology, Karolinska University Hospital Solna during this periodwere asked to participate. Patients were required to be older than 18years, otherwise there were no exclusion criteria. Population controls,individually matched to the first 322 SLE patients were identified inthe population registry. Matching was performed within one year of age,for sex, and region of living. Controls were contacted and asked toparticipate through a letter. The only exclusion criterion amongcontrols was a diagnosis of SLE. The Local Ethics Committee of theKarolinska University Hospital approved the study protocol. Allparticipants gave informed written consent to participate.

Data Collection.

All patients and controls were investigated in person by arheumatologist. Traditional risk factors for CVD were tabulated.Hypertension was defined as a systolic BP>140 mm Hg and/or a diastolicBP>90 mm Hg or use of antihypertensive treatment. Diabetes wasconsidered present if patients were previously diagnosed with diabetes.History of vascular events, defined as a history of objectively verifiedmyocardial infarction, ischemic cerebrovascular disease or peripheralvascular disease was obtained though interview and review of medicalfiles. In SLE patients, age at diagnosis, duration of disease, and lupusmanifestations including autoantibodies were recorded. Lupus nephritiswas defined according to the 1982 revised ACR classification criteriafor nephritis [Tan E M, et al. The 1982 revised criteria for theclassification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271-7]. Organ damage was assessed with Systemic Lupus InternationalCollaborating Clinics/ACR Damage index (SDI) [Gladman D, et al. Thedevelopment and initial validation of the Systemic Lupus InternationalCollaborating Clinics/American College of Rheumatology damage index forsystemic lupus erythematosus. Arthritis Rheum 1996; 39:363-9]. All bloodsamples were taken after overnight fasting and laboratory examinationswere performed blinded, either on fresh blood samples or after storagein −70° C.

Intima-Media Wall Thickness Measurements.

Three hundred and two patients were investigated with carotid ultrasoundusing a duplex scanner (Acuson 128XP, Mountain View, Calif., USA) with a7.0 MHz ART linear array transducer. Left and right carotids wereexamined. The IM thickness was defined as the distance between theleading edge of the luminal echo to the leading edge of themedia/adventitia echo [Wikstrand J, Wendelhag I. Methodologicalconsiderations of ultrasound investigation of intima-media thickness andlumen diameter. J Intern Med 1994; 236:555-9]. IM thickness was measuredover one cm length just proximal to the bulb. The mean intima-mediathickness (IMT) values for both sides were calculated for each subject.One technician recorded all measurements.

Determination of P45, P210 and β₂GPI Autoantibodies.

Peptides corresponding to the amino acids from 661 to 680 (P45;IEIGLEGKGFEPTLEALFGK, SEQ ID NO: 45) and amino acids 3136-3155 (P210;KTTKQSFDLSVKAQYKKNKH, SEQ ID NO: 210) of human ApoB100 were synthesized(K J Ross Petersen A S, Horsholm, Denmark) and used in ELISA. Thepeptides were modified by 0.5 M MDA for 3 h at 37° C. and dialyzedagainst PBS containing 1 mM EDTA as described [Fredrikson G N, et al.Identification of immune responses against aldehyde-modified peptidesequences in apo B-100 associated with cardiovascular disease.Arterioscler Thromb Vasc Biol 2003; 23:872-78]. Native and MDA-modifiedpeptides diluted in PBS pH 7.4 (20 μg/ml) were absorbed to microtiterplate wells (Nunc MaxiSorp, Nunc, Roskilde, Denmark) in an overnightincubation at 4° C. After washing with PBS containing 0.01% Tween-20(PBS-T) the coated plates were blocked with SuperBlock in TBS (Pierce,Rockford, Ill.) for 30 min at RT followed by an incubation of testplasma, diluted 1/100 in TBS-0.01% Tween-20 (TBS-T) for 2 h at RT andovernight at 4° C. After rinsing, deposition of autoantibodies directedto the peptide was detected using biotinylated rabbit anti-human IgM(ICN, Biomedicals, Inc., Aurora, Ohio) or IgG antibodies (Abcam, ab7159) appropriately diluted in TBS-T. After another incubation for 2 hat RT the plates were washed and the bound biotinylated antibodiesdetected by alkaline phosphatase conjugated streptavidin (BioLegend,405211), incubated for 2 h at RT. The colour reaction was developed byusing phosphatase substrate kit (Pierce) and the absorbance at 405 nmwas measured after 1 h of incubation at RT. Values are presented as theratio against a standard reference plasma. Data regarding thespecificity and variability of the antibody ELISA have been publishedpreviously [Fredrikson G N, et al. Identification of immune responsesagainst aldehyde-modified peptide sequences in apo B-100 associated withcardiovascular disease. Arterioscler Thromb Vasc Biol 2003; 23:872-78;Fredrikson G N, et al. Autoantibody against the amino acid sequence661-680 in apo B-100 is associated with decreased carotid stenosis andcardiovascular events. Atherosclerosis 2007; 194:e188-92].

Anti-β₂GPI antibodies IgM/IgG were determined with the multipleximmunoassays, BioPlex 2200 APLS (Bio-Rad Laboratories Inc., Hercules,Calif., USA). Results were reported in the ranges between 1.9-160 U/mlfor IgM and 1.9-160 U/ml for IgG. Results were handled as continuousvariables. The multiplex assays are regarded as positive if ≥20 U/ml.This cut-off level corresponded to at least the 99th percentile ofhealthy blood donors.

Statistics.

Clinical characteristics are presented as median (interquartile range,IQR) for continuous variables and as percentages for categoricalvariables. Continuous variables that were not normally distributed werelog transformed. If not normally distributed after log transformation,non-parametric tests were used. Depending on data type, Students't-test, Mann Whitney or Chi square test were used to compare differencesbetween groups. Correlations were investigated through calculating theSpearman rank correlation coefficients. Multivariable-adjusted logisticregression models were performed to evaluate the associations betweenautoantibodies and cardiovascular/organ damage outcomes. Partialcorrelations were calculated to determine the associations betweenautoantibodies and IMT controlling for age and sex.

The clinical characteristics of the SLE patient and control groups areshown in Table 24. Around 90% of the study subjects were females and themedian age was just below 50 years. The prevalence of clinicallymanifest CVD (myocardial infarction, stroke or peripheral arterydisease) was 13-fold higher in the SLE group.

TABLE 24 Clinical characteristic of systemic lupus erythematosus (SLE)patients and controls SLE patients (n = 434) Controls (n = 322) medianmedian (IQR)* (IQR)* P-value Age (years) 47.2 (34.2-57.8) 48.2(35.4-58.6) n.s. Female sex % 86 92 0.01 SLE characteristics Number ofSLE criteria 6 (5-7) 17 missing n.a. Disease duration year 10.6(2.8-20.9) n.a. SLICC damage index (SDI) 1 (IQR: 0-2, n.a. range 0-10)Traditional risk factors and laboratory tests Current smoking (%) 18.814.6 n.s. Systolic blood pressure (mmHg) 120 (110-140) 120 (110-135)n.s. Diastolic blood pressure (mmHg) 78 (70-80) 75 (70-82) n.s.Hypertension treatment (%) 37.2 13.7 <0.0001 Body mass index (kg/m2)24.0 (21.4-27.2) 24.3 (22.0-27.6) n.s. Diabetes (%) 1.4 0.9 n.s. Totalcholesterol (mmol/l) 4.9 (4.2-5.7) 5.1 (4.4-5.9) 0.009 High-densitylipoprotein (mmol/l) 1.1 (1.1-1.6) 1.5 (1.2-1.8) 0.006 Low-densitylipoprotein (mmol/l) 3.0 (2.5-3.6) 3.2 (2.6-3.9) 0.0002 Triglycerides(mmol/l) 1.0 (0.7-1.5) 0.78 (0.55-1.10) <0.0001 Apolipoprotein A1(mg/ml) 1.5 (1.3-1.7) 1.7 (1.4-1.9) <0.0001 Apolipoprotein B (mg/ml)0.81 (0.69-0.96) 0.81 (0.66-0.97) n.s. Glucose 4.8 (4.5-5.2) 4.9(4.6-5.2) n.s. High-sensitivity CRP 1.7 (0.7-5.3) 0.9 (0.4-0.9) <0.0001Creatinine 69 (58-84) 66 (59-73) 0.0005 Cardiovascular diseaseCardiovascular event† (%) 16.1 1.2 <0.0001 Ischaemic heart disease (%)6.5 0.3 <0.0001 Ischaemic cerebrovascular disease 8.7 1.6 <0.0001 (%)Ischaemic peripheral vascular 2.8 0.6 <0.0001 disease (%) IMT mm (meanof both sides) 0.053 (0.048-0.063) n.a. Treatment (ongoing) Prednisolone% 61.4 Anti-malarials % 37.1 Azathioprine % 17.4 Mycophenolate mofetil %11.4

For Table 24 above: *Distributions are given as median [interquartilerange (IQR)] unless indicated otherwise. ^(†)Includes myocardialinfarction, ischaemic cerebrovascular and peripheral artery disease.IMT=intima-media thickness; CRP=C-reactive protein; SLIC=Systemic LupusInternational Collaborating Clinics; n.a.=not applicable; n.s.=notsignificant.

SLE patients have lower levels of apo B P45 IgM and P210 IgG. We firststudied if there were differences in the expression of autoantibodiesagainst apo B between SLE patients and controls. This was determined byanalyzing IgM and IgG antibodies against the native and malondialdehyde(MDA)-modified apo B sequences P45 and P210. Autoantibodies againstβ₂GPI (also called apo H) were used to compare the pattern of apo Bpeptide autoantibodies with those against another antigen which binds tolipoproteins and to membrane phospholipids. SLE patients hadsignificantly lower levels of P210 IgG and P45 IgM (both the native andMDA-modified forms), while IgM against native and MDA-P210 wereincreased (Table 25).

TABLE 25 Apolipoprotein B and β₂-glycoprotein-I (GPI) autoantibodies insystemic lupus erythematosus (SLE) patients and controls. SLE patientsApolipoprotein B (n = 434) median Controls (n = 322) median antibodies(IQR)* (IQR)* P P45 IgM 0.64 (0.30-1.29) 0.86 (0.47-1.72) 0.001 MDA-P45IgM 0.72 (0.37-1.43) 0.92 (0.56-0.92) 0.001 P45 IgG 0.49 (0.23-1.03)0.42 (0.20-0.90) n.s. MDA-P45 IgG 0.52 (0.28-1.02) 0.43 (0.23-0.95) n.s.P210 IgM 0.77 (0.52-1.06) 0.70 (0.53-0.89) 0.007 MDA-P210 IgM 0.87(0.63-1.03) 0.79 (0.62-0.93) 0.002 P210 IgG 0.48 (0.24-0.84) 0.54(0.35-0.89) 0.02  MDA-P210 IgG 0.70 (0.51-0.98) 0.82 (0.61-1.05) 0.005b2GPI antibodies b2GPI IgM  1.9 (1.9-3.9)  1.9 (1.9-2.5) 0.002 b2GPI IgG 1.9 (1.9-10.2)  1.9 (1.9-1.9) 0.001

For Table 25 above: *Distributions are given as median [interquartilerange (IQR)]. Ig=immunoglobulin; MDA=malondialdehyde; n.s.=notsignificant.

Antibody levels against native peptides generally correlated stronglywith the level of antibodies against the MDA-modified form of the samepeptide but much more weakly with antibodies against the other apo Bpeptide. As an example, the Spearman correlation coefficient for P210IgG against MDA-P210 IgG was 0.85, while it was only 0.13 and 0.14 forP210 IgG against P45 IgG and MDA-P45 IgG, respectively. Similar trendswere also observed for P45 and P210 IgM. As expected, SLE patients alsohad significantly elevated levels of anti-β₂GPI IgG and IgM. The levelsof both P210 and MDA-P210 IgM correlated with β₂-GP-I IgG (r=0.19,P<0.001 and r=0.18, P<0.001; respectively) and b2-GP-I IgM levels(r=0.23, P=0.001 and r=0.21, P<0.01; respectively). The levels of P45and MDA-P45 IgM both correlated with β₂-GP-I IgM levels (r=0.13, P=0.001and r=0.14, P<0.001; respectively), but otherwise there were noassociation between autoantibody levels against apo B peptides andanti-β₂GPI. None of the common SLE medications were associated withautoantibodies against apoB, with the exception of antimalarials, whichwere negatively associated with MDA-P45 IgG (P=0.03).

Low levels of apo B P45 IgM and P210 IgG are associated with CVD in SLE.Next we compared autoantibody levels against apo B peptides and β₂GPI inSLE patients with and without prevalent CVD. CVD patients had lowerlevels of native and MDAP45 IgM, native P210 IgM, native and MDA-P210IgG, whereas β₂GPI IgG levels were higher. MDA-P45 IgM, MDA-P210 IgG andβ₂GPI IgG levels remained significantly different when adjusting for ageand sex (Table 26 and FIG. 14).

TABLE 26 Apolipoprotein B and β₂-glycoprotein-I (GPI) autoantibodies insystemic lupus erythematosus (SLE) patients with and withoutcardiovascular disease. CVD (n = 62) No CVD (n = 370) median (IQR)* SLEmedian (IQR)* P P adjusted Apo B antibodies P45 IgM 0.47 (0.19-1.17)0.70 (0.33-1.29) 0.01  n.s. MDA-P45 IgM 0.55 (0.24-1.02) 0.75(0.41-1.48) 0.003 0.04 P45 IgG 0.36 (0.17-0.90) 0.54 (0.23-1.05) n.s.n.s. MDA-P45 IgG 0.46 (0.29-0.84) 0.54 (0.28-1.04) n.s. n.s. P210 IgM0.64 (0.42-0.99) 0.78 (0.53-1.07) 0.03  n.s. MDA-P210 IgM 0.80(0.51-1.02) 0.54 (0.28-1.04) n.s. n.s. P210 IgG 0.32 (0.10-0.64) 0.50(0.25-0.88) 0.004 n.s. MDA-P210 IgG 0.60 (0.3-0.84) 0.72 (0.53-1.00)0.001 0.05 Apo H antibodies β₂GPI IgM  1.9 (1.9-3.5)  1.9 (1.9-4.0) n.s.n.s. β₂GPI IgG  4.0 (1.9-30.9)  1.9 (1.9-8.7) 0.02  0.01

Table 26 above: *Distributions are given as median [interquartile range(IQR)]. Ig=immunoglobulin; MDA=malondialdehyde; CVD=cardiovasculardisease; n.s.=not significant.

To further determine the association between these autoantibodies andcardiovascular disease in patients with SLE we used measurements ofcarotid IMT as assessed by ultrasonography. Associations were in anegative direction between carotid IMT and all apo B autoantibodies. ForP45, both native and MDA modified, IgM antibodies became significantafter age and gender adjustments, but these association were generallyso weak that the biological relevance is questionable. Crudeassociations for all P210 antibodies were significant, but theseassociations were not independent of age and sex (Table 27).

TABLE 27 Associations between apolipoprotein B and β₂-glycoprotein-I(GPI) autoantibodies and carotid intima-media thickness (IMT) in SLEpatients. SLE P adjusted for patients (n = 302) P age and genderApolipoprotein B antibodies P45 IgM −0.05 n.s. 0.02 MDA-P45 IgM −0.08n.s. 0.02 P45 IgG −0.10 n.s. n.s. MDA-P45 IgG −0.07 n.s. n.s. P210 IgM−0.23 0.001 n.s. MDA-P210 IgM −0.16 0.005 n.s. P210 IgG −0.20 0.001 n.s.MDA-P210 IgG −0.19 0.001 n.s. Apolipoprotein H antibodies β₂GPI IgM 0.04n.s. n.s. β₂GPI IgG −0.03 n.s. n.s.

Table 27 above: Partial correlations were calculated to determineindependent associations between autoantibodies and IMT when controllingfor age and sex. Ig=immunoglobulin; MDA=malondialdehyde; n.s.=notsignificant.

Low levels of apoB autoantibodies are associated with manifestation oforgan damage in SLE. Finally we determined if plasma levels of apo Bautoantibodies were associated with clinical signs of organ damage inSLE. SLE patients with permanent organ damage (SDI>1) had lower levelsof P45 IgM (both native and MDA-modified), P210 IgM and P210 IgG (bothnative and MDA-modified) than SLE patients with a SDI≤1 (Table 28). Whencontrolling for age and sex only the difference in MDA-P210 IgG remainedsignificantly different between the groups (Table 28). In contrast, SLEpatients with permanent organ damage had elevated levels of β₂GPI IgG(Table 28).

TABLE 28 Apolipoprotein B and β₂-glycoprotein-I (GPI) autoantibodies insystemic lupus erythematosus (SLE) patients with and without organdamage (SDI > 1). SDI > 1 median SDI ≤ 1 median P adjusted for (IQR)*(IQR)* P age Apolipoprotein B P45 IgM 0.53 (0.24-1.19) 0.72 (0.36-1.38)0.02  n.s. MDA-P45 IgM 0.60 (0.28-1.22) 0.79 (0.44-1.55) 0.003 n.s. P45IgG 0.48 (0.19-0.91) 0.50 (0.24-1.04) n.s. n.s. MDA-P45 IgG 0.50(0.30-0.97) 0.56 (0.27-1.03) n.s. n.s. P210 IgM 0.68 (0.44-1.02) 0.83(0.57-1.07) 0.006 n.s. MDA-P210 IgM 0.80 (0.55-1.02) 0.89 (0.68-1.05)n.s. n.s. P210 IgG 0.37 (0.16-0.66) 0.54 (0.28-0.95) 0.001 n.s. MDA-P210IgG 0.61 (0.41-0.84) 0.76 (0.56-1.05) 0.001 0.005 Apolipoprotein Hantibody β₂GPI IgM  1.9 (1.9-5.9)  1.9 (1.9-3.1) n.s. n.s. β₂GPI IgG 2.1 (1.9-21.5)  1.9 (1.9-7.6) 0.02  0.006

Table 28 Above: *Distributions are given as median [interquartile range(IQR)]. Ig=immunoglobulin; MDA=malondialdehyde; SDI=Systemic LupusInternational Collaborating Clinics damage index; n.s.=not significant.

Since many of the associations between antibody levels and organcomplications, including the cardiovascular, were found to be dependenton age we specifically analyzed the relationships between antibodylevels and age. All apo B autoantibodies were found to decrease with ageboth in SLE patients and in controls, while no such trend was observedfor β₂GPI antibodies (Table 29).

TABLE 29 Associations between apolipoprotein B and β₂-glycoprotein-I(GPI) autoantibodies and age in systemic lupus erythematosus (SLE)patients and controls. SLE patients Controls (n = 434) P (n = 322) PApolipoprotein B antibodies P45 IgM −0.19 0.001 −0.24 0.001 MDA-P45 IgM−0.22 0.001 −0.24 0.001 P45 IgG −0.17 0.001 −0.12 0.04  MDA-P45 IgG−0.16 0.001 −0.07 n.s. P210 IgM −0.26 0.001 −0.26 0.001 MDA-P210 IgM−0.19 0.001 −0.21 0.001 P210 IgG −0.29 0.001 −0.28 0.001 MDA-P210 IgG−0.30 0.001 −0.27 0.001 Apolipoprotein H antibodies β₂GPI IgM 0.07 n.s.0.12 0.04  β₂GPI IgG 0.01 n.s. 0.09 n.s.

For Table 29 above: Ig=immunoglobulin; MDA=malondialdehyde; n.s.=notsignificant.

Production of a multitude of autoantibodies is a characteristic featureof SLE and there is evidence that several of these autoantibodies, inparticular aPL, contribute to increased CVD in SLE. Autoantibodiesagainst the apo B-100 peptides P45 and P210 are found in mostindividuals and have on the contrary been associated with a lower CVDrisk in observational studies. The present study investigated how SLEaffects the occurrence of these potentially protective antibodies. Ourfindings demonstrate that subjects with SLE have reduced levels of P45IgM and P210 IgG. Moreover, SLE patients with clinically manifest CVDhad lower levels of P45 IgM and P210 IgG than those without CVD, butonly the levels of MDA-P45 IgM and MDA-P210 IgG remained significantlyafter controlling for age and sex. One possible explanation for thestronger association with autoantibodies recognizing the MDA-peptidescould be that these are more specific for epitopes present in oxidizedLDL. Also SLE patients with clinical manifestations of permanent organdamage as assessed by a SDI score>1 had lower levels of MDA-P210 IgG. Incomparison, anti-β₂GPI IgG levels were increased in SLE patients andthose with prevalent CVD had higher levels than those without. Takentogether these observations demonstrate that SLE is associated withsuppression of a set of naturally occurring autoantibodies withpotential protective effects and suggest that this may contribute toincreased risk for development of organ damage and CVD in SLE. There isevidence that some medications used to treat SLE also haveathero-protective effects. However, we found no association betweentreatment with prednisolone, azathioprine or mycophenolate mofetil andapoB peptide autoantibodies in the present study while the use ofantimalarias was associated with lower levels of MDA-P45 IgG.

There are several mechanisms through which autoantibodies to apoBantigens could protect against atherosclerosis and other types of organdamage in SLE. First, it is likely that such antigens are recognized bythe immune system first when LDL is modified by oxidation. Thisoxidation is associated with degradation of the apo B protein intosmaller peptide fragments as well as aldehyde-modifications.Aldehyde-modified apo B peptides are readily identified by the immunesystem but also non-modified apo B peptide sequences may be targeted bythe immune system if normally embedded into phospholipid LDL membrane.Oxidized LDL is cytotoxic for vascular cells and promotes theinflammation that leads to development and destabilization ofatherosclerotic plaques. Factors that facilitate an early removal ofoxidized LDL are therefore likely to have an athero-protective effect.Both P45 and P210 IgG have been shown to promote the uptake of oxidizedLDL in human monocyte/macrophages and treatment of LDLr^(−/−)/humanapoB^(+/+) mice with MDA-P45 IgG lowers the plasma level of oxidizedLDL. Oxidized LDL/MDAP45 immune complexes have anti-inflammatoryproperties through activation of the inhibitory FcγRII receptor.Treatment of hypercholesterolemic mice with recombinantmalondialdehyde-P45 IgG has been shown to inhibit development ofatherosclerosis and to promote plaque regression when combined withlowering of plasma cholesterol levels. Low levels of autoantibodiesagainst apoB peptides have been associated with more severeatherosclerosis and an increased risk for development of myocardialinfarction. Also IgM autoantibodies targeting phosphorylcholine (PC) inoxidized LDL have been attributed a protective role in cardiovasculardisease. Several studies have shown that subclinical carotid disease inSLE patients is associated with lower levels of these autoantibodiesadding further support to the notion that autoantibodies againstoxidized LDL antigens may protect against cardiovascular complicationsin SLE. In line with the notion that oxidized LDL contributes tovascular damage in autoimmune disease and that anti-PC antibodies mayprotect against this damage Ajeganova and coworkers reported thatdevelopment of cardiovascular events in rheumatoid arthritis isassociated with both elevated plasma levels of oxidized LDL and lowerlevels of anti-PC IgM.

β₂GPI is an evolutionary conserved protein, which occurs abundantly inthe human circulation. The function of β₂GPI is still underinvestigation, but recent data indicate that β₂GPI is mainly a scavengermolecule with capacity to bind and remove harmful bacterial productse.g. LPS. It is also involved in clearance of endogenous waste such asmicro particles and cellular debris. There is growing evidence that lowaffinity anti-β₂GPI, in similarity to some anti-apoB antibodies, belongto the natural antibody repertoire, which defends us againstwell-conserved pathogenic structures e.g. bacterial antigens or productsof oxidation. In most previous studies anti-β₂GPI antibodies areregarded as present or absent according to cut-offs used in the APScriteria. In this study, however, we have in similarity to anti-apoBantibodies investigated continuous titers and isotypes. Our resultsdemonstrate that only anti-β₂GPI antibodies of the IgG isotype occurredat higher titers in SLE patients as compared to controls, and hightiters are especially common in the SLE subgroup with previous CVD. Wealso note that, unlike anti-apoB antibodies, anti-β₂GPI antibodies donot decline with age, rather in the control group titers were higheramong older subjects.

Loss of tolerance against abundant apoptotic cell antigens is animportant pathogenic factor in SLE. In atherosclerosis the loss oftolerance against apoptotic cell antigens appears to take placeprimarily within the environment of the atherosclerotic plaque wherethere is a similar loss of tolerance against antigens in oxidized LDL.Taken together, these findings imply that the issue of tolerance controlmay be particularly critical in SLE atherosclerotic lesions. OxidizedLDL is enhanced in SLE and may further aggravate pro-inflammatoryresponses to apoptotic cells in SLE atherosclerotic lesions by competingwith the binding to phagocytic receptors, which mediate clearance ofboth apoptotic cells and oxidized LDL. It is likely that thesemechanisms play a role in the accelerated atherosclerosis in SLE andthat antibody-mediated removal of oxidized LDL could help to limitvascular and possibly also the general systemic inflammation in SLE.

We report that patients with SLE, a condition generally characterized bya high production of auto-antibodies, have reduced levels ofathero-protective autoantibodies against the apo B-100 peptides P45 andP210. The level of these antibodies was further reduced in SLE patientsthat had developed CVD. We propose that an impaired antibody-mediatedremoval of oxidized LDL may contribute to loss of tolerance andincreased inflammation in vascular tissues in SLE.

Example 5. Immunization with ApoB100 Peptide Vaccine ReducesAtherosclerosis Development in a Mouse Model of Systemic LupusErythematosus

In the present study we investigated if immunization with CVX-4, aprototype vaccine consisting of the apoB peptide P210, a bovine serumalbumin (BSA) carrier and the aluminum phosphate adjuvant Adjuphos,affects atherosclerosis development in MRL/lpr/ApoE^(−/−) mice thatdisplay both hypercholesterolemia and a SLE-like phenotype.

Experimental Methods

Mice:

Animal care and experimental procedures were approved by the localcommittee of Animal Care and Use at Lund University. MRL/lpr ApoE^(−/−)mice on a C57bl/6 background (originally from Jackson Laboratories,Charles River Laboratories, Germany) were bred in the animal facilityand female mice were used for the present study. High fat diet (HFD,0.15% cholesterol, 21% fat, Lantmännen, Sweden) was introduced from 12weeks of age until the experimental end point. Mice were givensubcutaneous injections (200 μL) of P210 conjugated to bovine serumalbumin (BSA, CVX-14) together with aluminium phosphate gel (Adjuphos)as adjuvant at 6, 9, 11 and 21 weeks of age. Injections ofphosphate-buffered saline (PBS) or Adjuphos alone served as controls.Mice were killed at 22 weeks of age by intraperitoneal injection ofketamine and xylazine. Spleens were harvested and stored in PBS on iceand plasma was collected by cardiac puncture and stored at −80° C. untilanalysis. Mice were then whole-body perfused with PBS followed byHistochoice (Amresco, Solon, Ohio, USA), and the descending aorta wasthen dissected free of connective tissue and fat, cut longitudinally,mounted en face and stored in Histochoice [Schiopu, A., et al. 2004.Recombinant human antibodies against aldehyde-modified apolipoproteinB-100 peptide sequences inhibit atherosclerosis. Circulation 110:2047-2052]. The hearts were collected and snap frozen in liquid nitrogenfor storage or sectioning.

Staining of the Descending Aorta:

En face preparations of the descending aorta were washed in distilledwater, dipped in 78% methanol and stained for 40 min in 0.16% Oil Red Odissolved in 78% methanol/0.2 mol L) 1 NaOH. The Oil Red O stainedplaque areas were quantified blindly using BioPix iQ 2.3.1 (BioPix AB).

Immunohistochemistry:

Frozen hearts were embedded in Tissue Tek (Sakura Fine Tek., Japan) andsections of 10 μm were cut from the aortic root for immunohistochemicalstaining of atherosclerotic plaques. Sections were fixed in ice-coldacetone for 10 minutes followed by permeabilized in 0.5% TritonX-100(Merck, Millipore, US) with PBS washing for 5 minutes between each step.Further, sections were blocked in 10% mouse serum in PBS for 30 minutes.Immunohistochemical staining of macrophages using rabbit anti-CD68(Abcam, Cambridge, UK) detected by secondary biotinylated goatanti-rabbit (Abcam) with diaminobenzene (DAB, Vector Labs, CA, USA) wasperformed. The primary or secondary antibodies were omitted as controls.Immune stained areas were quantified blindly in BioPix 2.0 Software(BioPix AB, Goteborg, Sweden).

Spleen Cell Preparation and Cell Culture:

Splenocytes in single cell suspension were prepared by pressing thespleen through a 70-μm cell strainer (BD Falcon, Franklin Lakes, N.J.,USA). Erythrocytes were removed using red blood cell lysing buffer(Sigma, St. Louis, Mo., USA). Cells were cultured in culture medium(RPMI 1640 medium containing 10% heat-inactivated foetal calf serum, 1mmol L⁻¹ sodium pyruvate, 10 mmol⁻¹ Hepes, 50 U penicillin, 50 μg/mLstreptomycin, 0.05 mmol L⁻¹ β-mercaptoethanol and 2 mmol L⁻¹L-glutamine; GIBCO, Paisley, UK) in 96-well, round-bottom plates(Sarstedt, Nümbrecht, Germany).

Flow Cytometry:

Splenocytes were stained with fluorochrome-conjugated antibodies andmeasured with a CyAn ADP flow cytometer (Beckman Coulter). The followingantibodies phycoerythrin/Cy7-conjugated anti-CD3, pacificblue-conjugated anti-CD4, allophycocyanin-conjugated anti-CD25,phycoerythrin-conjugated anti-Foxp3 and allophycocyanin/Cy7-conjugatedanti-CD8 were used for T cells and fluorescein isothiocyanate-conjugatedanti-B220, pacific blue-conjugated anti-CD11b,phycoerythrin/Cy7-conjugated anti-CD11c and allophycocyanin-conjugatedanti-CD115. The analysis was performed with FlowJo V10 software (TreeStar).

Measurements of IgG:

A 96-well ELISA plate was coated with 20 □g/ml P210 diluted inNa₂CO₃—NaHCO₃ and incubated at 4° C. overnight. After three cycles ofwashing, the plate was blocked with 2% goat serum in PBS for 60 minutes.After a single wash plasma samples diluted 1:50 was added to the plateand incubated for 1 hour in 37° C. After three washing cycles, detectionantibody (anti-mouse IgG-biotin) was added and the plate was incubatedfor 1 hour in 37° C. After three more washing cycles, streptavidin-HRPwas then added and incubated for 20 minutes in the dark. Stop solution(1M H₂SO₄) was then added and the absorbance was read at 450 nm.

Plasma Cholesterol and Triglyceride:

Total plasma cholesterol and triglyceride levels were quantified withcolorimetric assays, Infinity™ Cholesterol and Triglyceride (INT),respectively (Thermo Scientific).

Plasma Cytokines:

Cytokine (IL-2, IL-4, IL-5, IL-6 and IL-10) concentrations in plasmawere determined with multiplex technology (Luminex Assay, R&D SystemsInc.) according to the manufacturer's instructions.

Statistics:

Data are presented as mean±standard deviation. Student's two-tailedt-test was used for normally distributed samples and the Mann-Whitneyrank sum test for skewed data. Statistical significance was consideredat the level P<0.05.

MRL/lpr/ApoE−/− mice received 4 subcutaneous injections with PBS, CVX-14or adjuphos alone at week 6, 9, 11 and 21 of age. Mice were killed atweek 22 of age and atherosclerosis assessed by Oil Red O (ORO) en facestaining of the aorta and measurement of the cross-sectional plaque areaat the aortic root. Immunization with CVX-14 reduced plaque developmentin the aorta by 55.5% as compared to the PBS (1.32±0.36 versus2.97±2.26% ORO stained area, p=0.0133; FIG. 15A). A trend towardsreduced atherosclerosis was also observed in the mice give adjuphosalone. There was no significant difference in aortic root plaque areabetween the PBS and CVX-14 treated groups (FIG. 15B), but CVX-14treatment reduced the plaque area with positive macrophage staining(CD68) by 66.2% as compared with the PBS control (11.2±6.9 versus34.6±6.9%, p=0.005; FIG. 15C and FIG. 15D). There was no effect onplaque macrophage staining by giving adjuphos alone. The effect ofCVX-14 treatment on vascular inflammation was further assessed byanalyzing cytokine mRNA expression in the carotid artery. Arteries fromCVX-14 treated mice were found to have reduced expression of TNF-α andTGF-β mRNA, as well as of Foxp3 mRNA (FIG. 15E-FIG. 15G).

Plasma cholesterol was increased in CVX-14 treated mice compared to PBScontrol, whereas no difference was observed in triglyceride levels orbody weights (Table 30). There were also no significant differencesbetween P210 IgG1 between the Groups (Table 30).

TABLE 30 PBS CVX-14 Adjuphos Cholesterol 2.80 ± 2.22 4.77 ± 1.41 4.30 ±0.69 Triglycerides 0.63 ± 0.55 0.92 ± 0.21 0.99 ± 0.22 P210-IgG1 0.47 ±0.32 0.45 ± 0.32 0.51 ± 0.48

Splenocytes were analyzed with flow cytometry to determine systemicchanges of the immune system in immunized mice. There was an increasedfrequency of regulatory T cells, defined as CD3⁺CD4⁺CD25⁺Foxp3⁺ cells,in mice immunized with CVX-14 as compared to the PBS controls, but asimilar increase was also observed with Adjuphos alone (FIG. 16A).Immunization with CVX-14 also reduced the fraction of CD8⁺ T cells (FIG.16C), whereas no effect was seen on the fraction of splenic plasmacytoiddendritic cells and CD11b⁺CD115⁺ (FIG. 16D-FIG. 16E).

There is accumulating evidence that autoimmune responses againstmodified LDL particles aggravate arterial inflammation and developmentof atherosclerosis. As loss of tolerance against self-antigens is animportant feature of SLE and atherosclerosis it reasonable to assumethat autoimmune responses against LDL trapped in the vascularextracellular matrix contributes to the development of cardiovascularcomplications in SLE. In the present study we found that immunizationwith the ApoB peptide P210 reduces plaque development and inflammationin an atherosclerosis-prone mouse model of SLE. This effect wasassociated with reduced arterial cytokine expression and an expansion ofTregs in the spleen, whereas no change was observed in the levels ofP210 IgG. These observations are in good agreement with previous studiesdemonstrating that immunization with apoB peptide and oxidized LDLinhibits atherosclerosis through activation of Tregs and promotion ofLDL tolerance. An expansion of Tregs in the spleen and a trend towardsreduced atherosclerosis was also observed in mice injected with adjuphosalone. Athero-protective effects of aluminum-based adjuvants havepreviously been identified in several studies and have been suggested tobe explained by an uptake of oxidized LDL by the adjuvantsubcutaneously. Irrespective of this, aluminum-based adjuvants representattractive candidate adjuvants for possible future atherosclerosisvaccines.

There is a considerable unmet clinical need for improved prevention ofcardiovascular complications in SLE. Treatment with statins has notaffected the progression of carotid disease in randomized clinicaltrials involving subjects with SLE and it remains unclear to what extentother medications used in treatment of SLE reduces the cardiovascularrisk. In this context, oxidized LDL represents an interesting possibletarget as it has been implicated both in SLE and atherosclerosis.Autoantibodies against oxidized LDL-associated phospholipid antigens arecommon in SLE. Oxidized LDL and apoptotic cells compete for binding tothe same scavenger receptors on macrophages suggesting that presence oflarge amounts of oxidized LDL may further impair handling of apoptoticcells in SLE. Human atherosclerotic plaques contains T cells specificfor oxidized LDL antigens and T cells recognizing epitopes in apo B havebeen shown to accelerate atherosclerosis in experimental mouse models.Immunizations with the ApoB100 derived peptides could potentiallyrepresent an attractive approach not only for preventing damage to thecardiovascular system in SLE because it may also have effects on otherorgans. As previous studies have shown that immunizations with theApoB100 derived peptides promotes generation of LDL specific Tregs it islikely that these cells would have an immune-suppressive effect in anyorgan where they encounter native or oxidized LDL.

The results from this study show that immunizations with P210-containingvaccines reduce atherosclerosis development in Apoe^(−/−) mice with aSLE like phenotype. In accordance with previous studies usingP210-containing vaccines in hypercholesterolemic Apoe^(−/−) mice withoutSLE, regulatory T cells are increased systemically after the P210immunization. ApoB peptide based vaccines represents a possible novelapproach for prevention of CVD in SLE that warrants furtherinvestigation.

Example 6

Inflammatory responses in the autoimmune disorder systemic lupuserythematosus (SLE) results in severe clinical manifestations ofperipheral inflammation. Atherosclerosis, one inflammatory manifestationof the disease, arises as a result of lipid accumulation andmodification of lipoproteins in the arterial wall. Underlying bothmyocardial infarction (MI) and stroke, it is one of the main causes offurther cardiovascular disease (CVD) events and mortality. The incidenceof CVD is significantly increased in SLE patients suggesting that theenhanced systemic inflammation contributes to the dysfunctionalprotection against oxidized low-density lipoproteins (LDL) and otheratherogenic antigens. A vaccine formulation containing a peptidesequence of the LDL antigen was used to evaluate the effects ofatherosclerosis in a hypercholesterolemic SLE mouse to assess the immuneresponses. We assessed the atherosclerosis by examining plaquedevelopment in the aorta. The response to immunization was investigatedby flow cytometry of spleen and lymph node cells. Plaque progression inthe aorta was significantly decreased in the mice treated with thevaccine. Further, inflammatory immune cell populations weresignificantly decreased. This vaccine candidate represents as a possiblenew therapy against CVD in SLE for the future.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease inwhich the individual displays various immune responses against owntissue. (1) The inflammatory response given by this autoimmune conditionresults in severe clinical manifestations leading to complications suchas atherosclerosis, renal failure and hypertension amongst other things.

Atherosclerosis is a chronic inflammatory disease arising as a result oflipid accumulation and modification in the arterial wall. Underlyingboth myocardial infarction (MI) and stroke, it is one of the main causesof clinical manifestations of cardiovascular disease (CVD). Theatherosclerotic process is initiated when low-density lipoproteins (LDL)particles is entrapped in the vascular wall and modified by enzymes orreactive oxygen species forming oxidized LDL (oxLDL). Macrophagesattempt to remove oxLDL by engulfing them and eventually this leads tofoam cell accumulation, immune cell activation and arterial lesions inthe vessel wall.

The incidence of CVD is significantly increased in SLE patientssuggesting that the enhanced systemic inflammation contributes to thedysfunctional protection against oxLDL and other atherogenic antigens.For example, females with SLE have up to 50-fold increased risk ofgetting a MI, underlining the need for alternative preventives toinhibit atherosclerosis in SLE. Systemic autoimmune manifestations suchas SLE are suggested to go hand in hand with immunodeficiency. Thedeficiencies arise from defects of the immune system and featuresgenetic variations, environmental factors and immune cell activation.General characteristics of autoimmunity is antinuclear antibodies (ANA),anti-smith/ribonuclear proteins (anti-sm/RNP) and the ‘interferon (IFN)signature’, where peripheral blood cells display a significantupregulation of type I IFN-inducible genes.

Vaccines has been used a long time clinically to induce the individualsown immune protection against an antigen. Therefore, a vaccine throughantigen-specific modulation would be of great interest as a therapy. Weinvestigated whether immunomodulation therapies with the ApoB100 peptide45 are able to reduce atherosclerosis and apoptosis in experimental SLE.

Experimental Methods

Mice.

Female MRL/lpr ApoE^(−/−) mice (purchased from Jackson Laboratories,Charles River Laboratories, Germany) were bred in the animal facility.High fat diet (HFD, 0.15% cholesterol, 21% fat, Lantmännen, Sweden) wasintroduced from 6 weeks of age until the experimental end point. ApoB100peptide 45 (P45) was coupled to cholera toxin B (P45-CTB), as previouslydescribed (Sun J B, Czerkinsky C and Holmgren J. B lymphocytes treatedin vitro with antigen coupled to cholera toxin B subunit induceantigen-specific Foxp3(+) regulatory T cells and protect againstexperimental autoimmune encephalomyelitis. J Immunol. 2012; 188:1686-9).Treatments with 30, 15, 5 μg P45-CTB conjugate, 30 μg CTB or PBS startedat 18 weeks of age at day 0 and were orally administered at day 0, 2, 5,7, 14, 21, 28, 35, 42 and 49. Blood and urine was collected at day 0,14, 28, 42 and 56. Weight was monitored at day 0, 28 and 56. At day 56of treatment, the mice were killed by ketamin/xylazine injection at 26weeks of age. Blood for plasma was collected before a whole-bodyperfusion with PBS followed by collection of heart, kidney, carotids andmesenterial lymph node, which was snap frozen in liquid nitrogen andconsequently stored at −80° C. until analysis. Aorta was dissected freefrom surrounding tissues and fixed in Histochoice (Amresco, Solon Ohio,USA). Spleens and remaining mesenterial lymph node were collected andkept on ice until further analysis. Animal care and experimentalprocedures were approved by the local committee of Animal Care and Useat Lund University.

Lipid Staining of the Aortic Arch.

The aortic arch was prepared en face and washed in distilled water, 78%methanol and stained for 40 minutes in 0.16% Oil-Red-O dissolved in 78%methanol (0.2 mol/L NaOH). The quantification was performed blindlyusing BioPix i.Q 2.0 software (Biopix AB, Gothenburg, Sweden) wherebordeaux coloured regions were referred to the content of neutral lipidsin plaques.

Cytokine Determination of Spleen and Lymph Node Cells.

Spleens and lymph nodes were pressed trough a 70 uM single cell mesh,washed in RPMI and centrifuged 700×g for 5 minutes. Red blood cells fromspleens were lysed with Red Blood Cell Lysis buffer (Sigma). Cells werecalculated, washed in RPMI and centrifuged 700×g for 5 minutes followedby resuspension in cRPMI supplemented with 2% FBS and seeding at adensity of 0.5×10(6) cells/well. Cells were stimulated with CD3/28 beads(Life Technologies) and incubated for 72 h in 37*c, 5% CO2. The cytokinedetermination will be performed with a multiplex assay.

Flow Cytometry Analysis of Blood, Spleen and Lymph Node Cells.

Blood cells from day 42 were stained with CD3-PeCy7, CD4-PB, CD25-APC,FoxP3-PE IL-17-APC and IFN-γ-PE (Biolegend) for regulatory T-cell paneland T helper-1/T-helper 17 panel. Blood cells (20 μl) were also seededin a round bottomed 96-well plate in cRPMI supplemented with 10% FBStogether with 100 ug/ml Brefeldin A (Sigma Aldrich) for 24 h,consequently stained for flow cytometry analysis with CD3-PeCy7, CD4-PB,CD8-APC, IL-17-APC and IFN-γ-FITC (Biolegend) for T helper-1/T-helper 17panel.

Spleen and lymph node cells were stained for flow cytometry analysiswith anti CD3-PeCy7, CD4-PB, CD25-APC, FoxP3-PE, CD44-AF488, CD62L-PE,CD8-APC/Cy7, B220-FITC, B220-PB, CD24-PE, CD5-PE/Cy7, CD23-PB,CD21/35-APC/Cy7, CD1d-AF488, CD40-PE, mTGFβ-APC and CD86-APC/Cy7 (allBiolegend), run on Gallios Flow cytometer (Backman Coulter) and analyzedwith FlowJo software (Tree star).

Immunohistochemistry.

Frozen hearts and kidneys were embedded in Tissue Tek (Sakura Fine Tek.,Japan) and cross sections from the aortic root were collected with athickness of 10 and 5 μm, respectively. Macrophage, CD3 and CD8stainings were performed using rabbit anti-CD68 (Abcam, Cambridge, UK),rat anti-CD3 (Abeam, Cambridge, UK) and rat anti-CD8a (BD Parmingen,),respectively, and detected by diaminobenzene (DAB). Briefly, sectionswere permeabilized in 0.5% Triton X-100 (Merck Millipore, United States)and incubated in 3% H₂O₂ in PBS. Sections were blocked with 10% goat(CD68) or mouse (CD3 and CD8) serum (Sigma-Aldrich, United States) inPBS followed by incubation with 1 μg/ml of the primary anti-CD68, -CD3or -CD8 antibodies overnight at 4° C. in a pre-wet chamber.

Immunoglobulin G and M stainings of aortic arch and/or kidney will beperformed by using biotinylated anti-mouse IgG/IgM (Vector Laboratories)as primary antibodies, respectively. For detection of the anti-CD68, asecondary biotinylated anti-rabbit IgG (Vector Lab) produced in goat wasused and a biotinylated mouse anti rat (Vector Lab) for the detection ofCD3 and CD8. Color was developed with DAB Detection Kit (Vector Labs,CA, USA) binding to the biotinylated antibody and immune stained areaswere quantified in BioPix i.Q 2.0 software.

In Vitro Induction of Tregs Through Antigen Presentation

CD11c⁺ dendritic cells were isolated from a MRL/lpr ApoE−/− spleen usingCD11c positive selection kit and EasySep magnetic beads (StemCellTechnologies). CD4+ T cells were isolated using CD4 negative selectionkit (StemCell Technologies) followed by isolation of CD25⁻CD4⁺ cellswere isolated using CD25 positive selection kit (StemCell Technologies).Cells were cultured in complete RPMI (RPMI-1640 medium containing 2%heat-inactivated fetal bovine serum (FBS), 1 mmol L-1 sodium pyruvate,10 mmol L-1 Hepes, 50 U penicillin, 50 μg mL-1 streptomycin, 0.05 mmolL-1 b-mercaptoethanol and 2 mmol L-1 L-glutamine; all from GIBCO) forall experiments.

CD11c+ cells were pulsed with 5, 15, 30 μg/ml P45-CTB, 30 μg/ml CTB orPBS for two hours in 37° C. and thereafter washed three times in PBS.CD25⁻CD4⁺ cells were incubated with Celltrace Violet proliferationmarker (Thermo Scientific) for one hour in 37° C. 100 000 CD11c+ cellswere then cocultured with 100 000 CD4+CD25− T effector cells in completeRPMI for 72 hours. IL-2 and TGF-β (Peprotech) were added to all wells at25 U/ml and 10 ng/ml respectively, to induce Tregs. Dendritic cells andTreg induction was verified by flow cytometry using the antibodies antiCD123 PE, CD86-PeCy7, CD3-PeCy7, CD25-APC, FoxP3-PE, CD4-AF488,CD8a-APC/Cy7 as previously described above.

Previous studies using P45 in hypocholesterolemic mice have shown thatimmunization is associated with inhibited atherosclerosis progression aswell as eliciting a protective immunoresponse with anti-inflammatoryproperties. Moreover, there is clinical evidence of an associationbetween P45 IgG autoantibody levels and atherosclerotic plaqueinflammation, repair and cardiovascular events in endarterectomypatients. To investigate if the ApoB100 peptide 45 is able to induceatheroprotection and reduce apoptosis in experimental SLE, MRL/lprApoE^(−/−) mice were orally immunized with P45-CTB (5, 15 or 30 μg/ml),CTB (30 μg/ml) or PBS at treatment day 0, 2, 5, 7, 14, 21, 28, 35, 42and 49.

En face mounted aorta were analysed with Oil Red O (ORO) where theaortic arch demonstrated decrease of plaques with 5 and 15 ug/ml P45-CTBcompared to the PBS control (FIG. 17). Immunization with 5 and 15 μg/mlP45-CTB reduced the ORO positive staining for lipids with 58.1 and 58.5%respectively (12.1±5.0% and 12.1±4.1%, p<0.01 in both) compared to thePBS treated group (20.8±4.9%). However, no reduction observed in thegroup treated with 30 μg/ml P45-CTB compared to PBS. No significantdifference between each treatment group and CTB, although a trend ofreduction can be seen between 5 and 15 μg/ml P45-CTB compared to CTB.

The populations of immune cells in spleen and lymph node at the day ofsacrifice were influenced by the treatment, especially the groups of 5and 15 μg/ml P45-CTB. The central memory helper T cells(CD3⁺CD4⁺CD62L⁺CD44^(int-hi)) were reduced in spleen in the 15 and 30μg/ml P45-CTB groups compared to CTB (7.6±7.7% and 7.5±6.1% versus19.4±5.2% p<0.001 and p<0.01 respectively) but not PBS (FIG. 18A-FIG.18I). In lymph node, the central memory T cells were reduced in 15 μg/mlP45-CTB compared to CTB (18.8±11.5% versus 34.5±10.6%, (FIG. 19A-FIG.19I). Interestingly, the cytolytic T cell populations(CD3⁺CD8⁺CD62L⁺CD44^(int-hi)) in spleen displayed an increase in the 5and 15 μg/ml P45-CTB groups compared to both PBS (29.4±13.3% and31.9±15.1% respectively versus 17.0±5.1%, p<0.05 in both) and CTB(18.2±3.7%, p<0.05 in both). In contrast, a reduction was observed inlymph node for 15 μg/ml P45-CTB compared to PBS (23.0±16.3% versus41.3±7.6%, p<0.05 (FIG. 18A-18I, FIG. 19A-19I).

No differences in the amount of Tregs in spleen and LN were observed atthe day of sacrifice. However, staining blood cells for Tregsmid-treatment at day 42, a trend can be seen between the treatmentgroups and PBS and CTB controls where an slight increase can be observedin the 5 and 15 μg/ml P45-CTB groups, although not significant.

Previous observations suggest there is a possibility to develop anApoB100 peptide-conjugated vaccine targeting atherosclerosis. SLEpatients has a significant increased incidence of CVD as a result ofsystemic inflammation and thus in need of specific therapies. In thisstudy provide data that an ApoB100 peptide (P45) coupled to CTB haveimmune-modulating properties and reduces atherosclerosis in the aorticarch in MRL/lpr ApoE^(−/).

Plaque formations in the 5 and 15 μg/ml P45-CTB treatment groups weredecreased compared to PBS control, suggesting atheroprotection iselicited by the vaccine candidate in lower doses. The immune cellpopulations seen in spleen and lymph node reveals how the vaccineinfluence the immune system, thus there were differences in the T cellpopulations. Central memory helper T cells were reduced in spleen aftertreatment with 5 and 15 μg/ml P45-CTB compared to CTB. In the lymphnode, the effects were not as pronounced, only a reduction in the 15μg/ml P45-CTB group was seen. The activated central memory cytolytic Tcells in spleen were increased in the 5 and 15 μg/ml P45-CTB treatmentgroups and similarly in the lymph node. This might suggest an expansionof the CD8⁺ portion of T cells resulting in less CD4⁺ cells in theperiphery after exposure to peptide vaccine. Immune protection wouldrather be elicited through cytolytic T cell activation from antigenspecificity initiated by the vaccine rather than regulatory T cellsuppression, even though a slight trend in populations can be seen.

Taken the results from Oil Red O and the flow cytometry immune cellpopulation characteristics together, it is distinct that an effect ofthe immunizations with the vaccine candidate in lower doses is observed,either compared to PBS and/or to CTB. As seen in our previous studiesusing P45-CTB as a therapy against atherosclerosis, we can now seeimmunomodulating effects in an SLE setting as well. This peptide vaccinerepresents a possible new therapy for treatment of CVD in SLE patients.

Example 7. Passive Immunization with Recombinant Human IgG1 Against anApoB100 Peptide Decreased Atherosclerosis Development in a Mouse Modelof SLE

Background.

Systemic lupus erythematosus (SLE) in an autoimmune diseasecharacterized by inflammation in several organs, such as blood, joint,kidneys, skin and the nervous system. In SLE, increased generation andimpaired clearance of apoptotic material is believed to be one diseasepromoting mechanism leading to activation of autoimmune cells. SLEpatients have an increased risk of cardiovascular disease (CVD) (e.g.,an about 50-fold increased risk of myocardial infarction) and CVD is themain cause of death in these patients. Traditional preventive CVDtherapies such as statins do not lower the incidence of CVD in SLEpatients stressing the fact that novel therapies targeting other diseasemechanisms is needed. Patients suffering from SLE typically haveincreased Th1, Th17 and CD4⁻CD8⁻ cells; decreased CD8+ cells and Tregs,more reactive T effector cells; and decreased activation threshold.

Aim.

To test if orticumab (BI-204; a recombinant human IgG against peptidesequence 45 in ApoB100) reduced atherosclerosis development in a mousemodel of SLE.

Results.

SLE ApoE^(−/−) mice were treated with three weekly injections of eitherorticumab or an irrelevant antibody as control. Six-week old mice werefed with HFD, and were injected at weeks 18, 19 and 20 with orticumab(one dose of 1 mg per week, IP injections), and sacrificed for analysisin week 22. Plaque area and composition were determined in the aorticroot. Mice treated with orticumab had significantly smaller plaques withless macrophages (FIG. 25A). Flat preps of the aorta showed a reduction,although not significant, in lipid staining after treatment withorticumab (FIG. 25B). Orticumab-treated mice also had a reduced CD68+area compared to mice treated with a control antibody (FIG. 26). Therewas also increased apoptosis in plaque areas in orticumab-treated miceas quantified by the number of TUNEL cells (FIG. 27). There were nodifferences in spleen monocytes or B cells at the end of experiment.Furthermore, no differences in plasma levels of oxidized LDL and plasmacytokines could be detected (FIG. 28).

Conclusions.

Orticumab treatment in SLE ApoE mice has the potential to reduceatherosclerosis development.

Example 8. Phase I Study: Double-Blind, within-Group RandomizedDose-Escalation Trial Evaluated Single and Multiple Doses of Orticumab

Study population: 80 healthy subjects with elevated LDL cholesterol.

Orticumab was provided as a sterile liquid and contained nopreservatives. Each single-use, 10 mL vial was designed to deliver 250mg of MLDL1278A. The Drug Product was formulated as 25 mg/mL orticumabin 20 mM sodium acetate, 0.15 M sodium chloride, pH 5.5.

Route of Administration: intravenous injection (Subcutaneous injectionwas also examined).

Overall, the data obtained from single- and multiple-dose IVadministration exhibited a clearance (CL) of approximately 8.96-12.1mL/kg/day, which is higher than the CL of a typical IgG1 antibody(typical CL, 2-5 mL/kg/day). See FIG. 29. The terminal phase volume ofdistribution (Vz, 216-478 mL/kg) was also higher than that of a typicalIgG1 antibody. The exact explanation for these unusual PKcharacteristics was unknown at that time. The mean terminal half-lifewas similar after both single and multiple administrations of orticumaband was generally in the range of 2-3 weeks and thus in agreement withother therapeutic monoclonal antibodies. The dose-normalized AUCinf, CL,volume of distribution, and terminal elimination half-life were similarafter single and multiple IV doses, which indicated that the PKparameters did not change over time.

Safety: There were a total of 151 treatment-emergent adverse events. Themajority of these (125; 82.8%) were mild in intensity; the rest (26;17.2%) were moderate; none were severe. Of the 151 adverse events, 115(76.2%) were reported by 44 of the 64 subjects on active treatment,whereas 36 (23.8%) were reported by 17 of the 24 subjects treated withplacebo.

Efficacy (effect of orticumab on Biomarkers): The Phase I studyinvolving healthy volunteers did not include any direct efficacyendpoints, but a panel of biomarkers and lipids was evaluated. The panelincluded: oxLDL, Lp-PLA2, hsCRP, sCD36, MCP-1, ICAM-1, IL-10, VCAM-1,IL-1ra, TNFα, insulin, and MMP-3. No treatment-induced general effectsof MLDL1278A were observed in these biomarkers.

Example 9. Second Phase I: Open-Label, Single-Dose Bioavailability Studyof Orticumab after Subcutaneous and Intravenous Administration

Primary Objective:

to estimate the absolute systemic bioavailability of 150 mg/mL orticumabadministered via subcutaneous (SC) injection. Secondary Objectives: toexplore the possible effect of a prior intravenous (IV) dose on SCbioavailability; to further characterize the pharmacokinetics (PK) oforticumab; to further evaluate safety and tolerability following SC andIV administration of orticumab; to further characterize theimmunogenicity of orticumab by measuring antibodies to orticumab.

Results

Following SC administrations of a single dose of 360 mg orticumab inCohort A, Period 2 and Cohort B, the mean serum concentrations slowlyreached a maximum in approximately 4 days (reaching 15.1 and 9.15 μg/mL,respectively). See FIG. 30. The absolute bioavailability of orticumabafter a single SC dose of 360 mg orticumab, based on a statisticalanalysis of AUC(0-inf), was 47%. The bioavailability of orticumab aftera single SC dose of 360 mg orticumab, which was administered after atleast 70 days washout from an initial IV dose, was 56%. Therefore, aprior IV dose slightly increased (approximately 9%) the bioavailabilityof the subsequent SC dose. Orticumab was slowly absorbed after a singleSC dose with a median tmax of 4 days. Orticumab t½ was about 20 days forboth routes of administration.

There were no SAEs during the study and no subjects were withdrawn as aresult of AEs. The most frequently reported TEAEs overall were mildintensity local site reactions following the SC injections (and similarin both groups, SC+/IV).

TABLE 31 Summary of pharmacokinetic parameters of orticumab. IntravenousSubcutaneous (Cohort A, (Cohort A, Subcutaneous Summary Period 1) Period2) (Cohort B) Parameters Statistics n = 12 n = 12 n = 9 AUC_((0-inf))Mean (SD)  412 (87.3)  236 (69.7)  194 (45.6) (μg * d/mL) AUC_((0-last))Mean (SD)  380 (76.4)  215 (60.0)  178 (39.6) (μg * d/mL) C_(max)(μg/mL) Mean (SD) 84.3 (16.9) 15.1 (4.50) 9.15 (3.86) t_(max) (d) Median(min-max)    0.04 (0.04-0.04)    3.99 (1.96-6.91)    3.91 (1.00-4.02)t_(1/2) (d) Mean (SD) 20.3 (2.91) 20.5 (2.84) 19.6 (6.03) MRT (d) Mean(SD) 16.9 (3.02) NA NA CL (L/d) Mean (SD) 0.906 (0.168) NA NA CL/F (L/d)Mean (SD) NA  1.65 (0.491)  1.96 (0.522) V_(ss) (L) Mean (SD) 15.2(3.25) NA NA V_(z) (L) Mean (SD) 26.3 (5.34) NA NA V_(z)/F(L) Mean (SD)NA 48.4 (14.6) 53.6 (15.4) min = minimum; max = maximum; NA = notapplicable; SD = standard deviation. Notes: Value of AUC(0-inf) inCohort A Period 2 was corrected by subtracting the AUCresidual left fromCohort A Period 1.

Example 10. Phase IIa Study: Double-Blind, Placebo-Controlled,Randomized, Multicenter Study Involving Patients on Standard-of-CareTherapy for Atherosclerotic Cardiovascular Disease (CVD) with Evidenceof Vascular Inflammation

Overview:

Evidence of vascular inflammation was quantified by [18F]2-deoxyglucosepositron emission tomography (FDG-PET) and computed tomography (CT).

The study enrolled 147 participants (83% male; mean age 63.0±9.0 years)with atherosclerosis and evidence of carotid or aortic plaqueinflammation, measured by FDG-PET/computed tomography (CT). Becausethere is poor correlation between serum LDL levels and both serum andplaque oxLDL levels, patients were enrolled with a relatively wide rangeof serum LDL levels (fasting LDL>60 mg/dL at screening, which includespatients both above and below National Cholesterol Education ProgramAdult Treatment Panel III treatment goals). 139 patients completed thestudy.

All patients were on standard-of-care therapy that included3-hydroxy-3methyl-coenzyme A (HMG-CoA) reductase inhibitor (statin). Thedose and type of statin was unchanged for at least 6 weeks prior toinitiation of screening, and patients remained on statin therapy withoutchange to dose or type of statin throughout the study. If patients werereceiving antihypertensive, antiplatelet agents, non-statinlipid-modifying agents, inhaled corticosteroids, leukotriene-modifyingagents, or thiazolidenediones, those therapies were also kept stable for6 weeks prior to screening and for the duration of the study. Allpatients who finished the study were treated with standard therapy.

A minimum of 10 patients with type 2 diabetes mellitus (T2DM) per arm(38 total) enrolled, to allow exploration of an effect oninsulin/glucose homeostasis.

Trial Design (FIG. 31):

Patients were randomly allocated in a 1:1:1 ratio to receive eitherMLDL1278A single infusion (1245 mg intravenously [IV]×1, followed byplacebo infusions to maintain blinding), MLDL1278A multiple infusions(1245 mg IV followed by 830 mg IV weekly×3 followed by 830 mg IVmonthly×2), or placebo by IV infusions to maintain blinding. Randomallocation was stratified by T2DM status and pre-diabetes status,presence of elevated screening tissue-to-background ratio TBR>2), andstudy site.

The study consisted of a 28-day screening period, a 78-day treatmentperiod, and a 90-day follow-up period. Study assessments that coincidedwith study treatment days were to be performed prior to dosing unlessotherwise specified. During the screening period, patient acceptabilityfor the study was assessed on the basis of medical history, concomitantmedications, physical examination, and clinical laboratory test results.Once a patient passed this initial screening phase, acceptability forstudy participation was confirmed on the basis of a TBR>1.8 in thequalifying vessel, as quantified by FDG-PET/CT. All FDG-PET/CT data wereacquired on combination FDG-PET/CT scanners; the CT data were used foranatomic reference and localization.

Demographic and selected baseline characteristics for thesafety-evaluable population, defined as all patients who received anydose of study drug, are presented in Table 32. The majority of patientswere male (83.7%) and White (89.7%). The average age was 63.3 years(range, 40-80 years). All treatment groups were similar with respect toage, sex, and race. The orticumab single-dose group had higherpercentage of patients with prediabetes (41.2%) compared with theorticumab multiple-dose (32.7%) and the placebo (27.7%) groups. Alltreatment groups were similar with respect to the other selectedbaseline characteristics.

TABLE 32 Demographic and baseline characteristics, safety evaluablepatients. (MLDL1278A refers to orticumab) MLDL1278A Multiple PlaceboSingle Dose Dose All Patients Characteristic (n = 47) (n = 51) (n = 49)(n = 147) Age, in years n 47 51 49 147 Mean (SD) 63.9 (8.9)   62.5(8.6)   63.3 (9.5)   63.3 (9.0)   Median   65.0   63.0   66.0   64.0Range 41-80 40-80 43-80 40-80 Sex, No. (%) of patients n 47 51 49 147Female  8 (17.0)  9 (17.6)  7 (14.3) 24 (16.3) Male 39 (83.0) 42 (82.4)42 (85.7) 123 (83.7)  Race, No. (%) of patients n 47 50 49 146 Asian 1(2.1) 0 (0.0) 1 (2.0) 2 (1.4) Black  5 (10.6) 4 (8.0) 4 (8.2) 13 (8.9) White 41 (87.2) 46 (92.0) 44 (89.8) 131 (89.7)  Baseline body mass index(kg/m²) n 47 51 49 147  Mean (SD) 29.4 (4.6)   29.2 (4.9)   29.1 (3.5)  29.2 (4.4)   Median   29.1   29.0   29.5   29.1 Range 21.7-49.319.3-38.8 20.4-36.9 19.3-49.3 Type 2 diabetes mellitus, no. (%) ofpatients n 47 51 49 147  Yes 14 (29.8) 12 (23.5) 12 (24.5) 38 (25.9) No33 (70.2) 39 (76.5) 37 (75.5) 109 (74.1)  Prediabetes, no. (%) ofpatients n 47 51 49 147  Yes 13 (27.7) 21 (41.2) 16 (32.7) 50 (34.0) No34 (72.3) 30 (58.8) 33 (67.3) 97 (66.0) Potent Statin use, no. (%) ofpatients n 47 51 49 147  Yes 24 (51.1) 23 (45.1) 21 (42.9) 68 (46.3) No23 (48.9) 28 (54.9) 28 (57.1) 79 (53.7) LDL-C n 47 51 49 147  Mean (SD)96.6 (35.0)   91.9 (20.8)   98.2 (29.8)   95.5 (28.9)   Median   93.0  95.0   95.0   94.0 Range  39.0-231.0  55.0-130.0  52.0-189.0 39.0-231.0

Primary Outcome Measures:

The primary outcome variable was change in index vessel MDS-TBR (mostdiseased segment-target to background ratio) measured by FDG-PET/CT frombaseline to week 12. Arterial FDG uptake was assessed as targetto-background ratio (TBR). Index Vessel was defined as the vessel withthe highest mean-of-the-maximum TBR and acceptable image quality atbaseline. The most diseased segment was defined as the vessel segment (3contiguous slices) that included the highest TBR at baseline.

Secondary Outcome Measures:

Incidence and severity of adverse events and clinical laboratoryabnormalities as a measure of safety and tolerability of orticumab.Effects of orticumab on inflammatory and metabolic biomarkers. Serum andEDTA plasma samples for soluble biomarker analysis were obtained atbaseline and on Day 29 and Day 85. Biomarker assays were performed usingthe HumanMAP1.6 multiplex panel from Rules Based Medicine (RBM). IL-6and TNFα immunoassays were performed at Pacific Biomarkers usingQuantikine R&D High Sensitivity ELISA. IL-10, MPO and oxLDL immunoassayswere performed at Pacific Biomarkers using immunoassay kits from MSA,Prognostix, and Mercodia, respectively. Presence of anti-therapeuticantibodies to orticumab.

Results:

MLDL1278A Did Not Reduce Either Imaging or Circulating Markers ofInflammation. Although high serum concentrations of orticumab wereachieved throughout the study for both treatment groups, treatment didnot significantly reduce arterial inflammation (primary endpoint ofindex vessel MDS-TBR) versus placebo. Baseline demographics werecomparable among groups for the 117 patients with evaluable week 12FDG-PET images included in the primary analyses. Orticumab was welltolerated, and there was no evidence of immunogenicity. Notably, anominal increase in the levels of tumor necrosis factor alpha (p=0.03)and interleukin 6 (p=0.04) occurred at 4 weeks in the multiple-dosegroup. No significant decreases in lipid parameters or high-sensitivityC-reactive protein level were observed in either group. Further, none ofthe secondary outcomes were different between groups. No serious adverseevents were reported that were related to the procedures or to theadministration of orticumab in this Study. This Study did notdemonstrate any clinically significant safety signals for orticumab, andthe only imbalances in safety events between dose arms were minornumerical differences in dizziness, headache, and gastrointestinal(diarrhea) events, which were assessed as mild and not clinicallysignificant by the Internal Monitoring Committee. No dose-limitingtoxicities, deaths, or pregnancies were reported. No trends inlaboratory values and significant clinical events were observed.

Example 11. Simulation and Actual Pharmacokinetics (PK)

The ability of orticumab to shut down macrophage pro-inflammatoryactivity locally in the plaque became the focus, as it was thought to bean important mechanism underlying its therapeutic activity. Initialhypothesis of using orticumab related to neutralizing oxLDL from thesystemic compartment, which required 4 μg/mL=˜28 nM=11 mg of orticumabto neutralize 90% systemic oxLDL. Further in vitro assays provided aninsight into the minimum effective concentration of orticumab needed toachieve 50% or 90% inhibition of oxLDL-mediated cytokine release (i.e.,inhibition of monocyte chemoattractant protein 1, MCP-1):

IC₅₀ for MCP-1 inhibition: ˜10 nM=1.5 μg/mL=˜4 mg

IC₉₀ for MCP-1 inhibition: ˜30-80 nM=4.5-12 μg/mL=12.4-33 mg.

The dosage (an initial dose of 1245 mg, followed by 830 mg weekly×3,then 830 mg monthly×2) in the clinical trial, compared to the lowerrange (12.4 mg orticumab) as IC₉₀ for MCP-1 inhibition, was 100 timesand 67 times greater. The dosage in the clinical trial, compared to thehigher range (33 mg orticumab) as IC₉₀ for MCP-1 inhibition, was 38times and 25 times greater.

In atherosclerosis patient, it has been reported that there is anincreased endothelial permeability to macromolecules, where uptake ofinjected macromolecules into the arterial wall is rapid and linear overtime and the equilibrium against circulating blood stream is reachedwithin 1 hour (Ross et al NEJM 1999).

Based on this background information, we proposed a loading dosage thatwould yield steady state plasma concentration of orticumab of at least12 μg/mL for up to 96 hours in a simulation model (FIG. 32). The loadingdose of 8 mg/kg (664 mg for an averaged patient of 83 kg), followed bybi-weekly to weekly 2 mg/kg (166 mg) subcutaneous (SC) dosing. Thisloading dose was 1.9 times less than the loading dose, and the weeklydosing was 5 times less than the weekly dosing.

In another set of simulations based on SC dosing:

(1) Weekly dosing at 2 mg/kg (166 mg) achieved 12 μg/mL thresholdbetween Day 2-4 (FIG. 33A);

(2) Biweekly dosing at 2 mg/kg did not achieve 12 μg/mL threshold, butdid achieve 4 μg/mL threshold from Day 1-6 (FIG. 33B);

(3) Monthly dosing at 2 mg/kg did not achieve sustained exposure at the4 μg/mL threshold (FIG. 33A);

(4) Loading Dose of 5 mg/kg (415 mg), followed by biweekly dosing at 2mg/kg did not achieve 12 μg/mL threshold, but did maintain exposureabove the 4 μg/mL through Day 6 (FIG. 33B).

In clinical trials of orticumab, Tables 33-36 below summarize the PKdata.

TABLE 33 The PK data from a “FIH” trial with single dosing. Dose Routeof C_(max) C ≥ C ≥ (mg/kg) Admin (μg/mL) 1 μg/mL C ≥ 4 μg/mL 12 μg/mL1.25 SC 2.99 Day 28 Not achieved Not achieved 1.25 IV 20.6 Day 28 Day 3(<Day 7) 11 hours 5 IV 105 Day 56 Day 14 Day 3 15 IV 286 Day 70* Day 42Day 28 (*Last day of assessment)

TABLE 34 The PK data from a “FIH” trial with multiple dosing. Dose Routeof C_(max) C ≥ C ≥ (mg/kg) Admin (μg/mL) 1 μg/mL C ≥ 4 μg/mL 12 μg/mL1.25 SC 1.24 Day 42 Not achieved Not achieved 1.25 IV 29.6 Day 70 Day 28Day 23 5 IV 105 Day 91* Day 56 Day 35 15 IV 303 Day 91* Day 91 Day 56(*Last day of assessment)

TABLE 35 The PK data from a “Ph1” trial with single dosing comparingsubcutaneous (SC) and intravenous (IV) administration. Dose Route ofC_(max) C ≥ C ≥ (mg) Admin (μg/mL) C ≥ 1 μg/mL 4 μg/mL 12 μg/mL 360 SC9.15 Day 43 Day 15 Not achieved 360 SC 70-90 days 15.1 Day 57 Day 15 Day8 after IV dosing 360 IV 84.3 Day 57* Day 15 Day 8 (*Last day ofassessment)

TABLE 36 The PK data from a “Ph2” trial. Dose Route of C_(max) (μ/mL) C≥ 1 C ≥ 4 C ≥ 12 Group Admin D 1 D 78* μg/mL μg/mL μg/mL Single IV 297N/A Day 106 Day 50  Day 22  Dose Multi- IV 286 197 Day 169 Day 141 Day106 ple Dose Dosing days: D 1, 8, 15, 22, 50, 78. *Last day of dosing.C_(max), Single Dose at 1.25 mg/kg: 1.25 mg/kg=104 mg, which was 6.9times greater max exposure with IV vs SC admin (½-life is 20 days forboth); SC dose does not reach threshold of 4-12 μg/mL.C_(max), Single Dose at 360 mg: 360 mg=4.34 mg/kg (3.5 times FIH study),9.2 times greater max exposure with IV vs SC admin (½-life is 33.5 vs24.3 days, IV vs SC); SC dose falls within range of 4-12 μg/mL.

As a result, based on the simulated and actual PK data, 8 mg/kg (664 mg)is optimal, but 5 mg/kg (415 mg) is likely sufficient. Based on the½-life of a single dose in the clinical studies (i.e., 24 days with asingle SC dose of 360 mg), monthly dosing is reasonable.

In another set of studies of orticumab:

(1) Weekly SC dose of 1 mg/kg and above maintained concentrations above4 μg/mL at steady state. FIG. 34 depicts the simulated human PK profilesafter weekly SC dosing, using PK parameters from Phase I data.Bioavailability after SC dose is 70%.

(2) Bi-weekly SC dose of 1.5 mg/kg and above maintained concentrationsabove 4 μg/mL at steady state. FIG. 35 depicts the simulated human PKprofiles after bi-weekly SC dosing, using PK parameters from Phase Idata.

(3) Monthly SC dose of ˜3 to 4 mg/kg and above maintained concentrationsabove 4 μg/mL. FIG. 36 depicts the simulated human PK profiles aftermonthly SC dosing, using parameters from Phase I data.

Based on simulated PK data, and targeting a plasma concentration of 12μg/mL threshold for maximum chance of success, weekly dosing should beno less than 2 mg/kg (166 mg); 4 mg/kg (332) would be preferable;biweekly dosing must be >2.5 mg/kg (208 mg); higher doses were notsimulated; and monthly dosing should be 6 mg/kg (498 mg). In someembodiments, final recommendation is 500 mg, monthly via SC injection.Monthly dosing of 500 mg each month can be administered 12 doses over a12-month dosing regimen, or 3 doses over a 3-month dosing regimen.Monthly dosing of 330 mg each month (to meet the minimum 4 μg/mL plasmaconcentration threshold) can be administered 12 doses over a 12-monthdosing regimen, or 3 doses over a 3-month dosing regimen.

Example 12. Immune Responses Against Oxidized LDL as Targets forPrevention of Atherosclerosis in Systemic Lupus Erythematosus

Abstract: Patients suffering from systemic lupus erythematosus (SLE) areat an increased risk of developing cardiovascular disease (CVD) andtraditional therapies including statins provide insufficient protection.Impaired removal of apoptotic material is a common pathogenic mechanismin both SLE and atherosclerosis and is considered to be a key factor inthe development of autoimmunity. We aimed to investigate if targetingoxidized LDL autoimmunity can affect atherosclerosis in SLE. Subjectswith SLE and matched healthy controls had similar levels of oxidized LDLin plasma but the correlation with IL-6 was stronger in SLE subjects.Mucosal immunization of hypercholesterolemic mice with an SLE-likephenotype (B6.lpr.ApoE−/− mice) with the apolipoprotein B-100 peptideP45 (amino acids 661-680) coupled to the cholera toxin B-subunitincreased regulatory T cells in mesenteric lymph nodes and reducedplaque development in the aorta by 32.5%. Targeting oxidized LDL byinjection of an MDA-P45 specific antibody reduced aortic atherosclerosisby 42.8%, subvalvular plaque area by 50.3% and the macrophage content ofremaining plaque by 30.5%. This exemplary study provides experimentaland clinical support for targeting oxLDL in the prevention ofcardiovascular complications in SLE.

INTRODUCTION

Accumulation and oxidation of low density lipoproteins (LDL) in thearterial wall is considered to be a pathogenic process of key importancein the development of atherosclerosis. Oxidized LDL (oxLDL) is stronglypro-inflammatory and has toxic effects on vascular cells. It can beremoved by infiltrating monocytes that differentiate into scavengerreceptor-expressing macrophages that ingest the oxLDL particles andstore excess cholesterol in lipid droplets. These cells may also presentantigens derived from oxLDL to T cells. Activation of Th1 cells resultsin aggravation of inflammation and progression of atherosclerosis, whileactivation of regulatory T cells (Tregs) has the opposite effect. Topromote and/or mimic the protective oxLDL immunity, we and others havedeveloped apolipoprotein B-100 peptide immune-suppressive vaccines andantibodies that reduce atherosclerosis in different mouse models.

Systemic lupus erythematosus (SLE) is a complex autoimmune diseasecharacterized by autoimmune responses against a number of differenttissues. The incidence of cardiovascular disease (CVD) is significantlyincreased in SLE patients with a 5-10 fold increased risk of myocardialinfarction (MI) after adjusting for the Framingham Risk Factors. Thepathogenic mechanisms responsible for the increased CVD risk in SLEremains to be fully understood. There is evidence that a dysfunctionalclearance of autoreactive lymphocytes and apoptotic material in SLE isan important cause of autoimmunity in SLE. Similarly, an impairedclearance of apoptotic material contributes to the development ofatherosclerosis. Macrophage scavenger receptors are of criticalimportance for the clearance of both apoptotic material and oxLDLindicating that both processes could be affected in SLE and that thiscan represent a link between SLE and CVD. We hypothesize that impairedremoval of oxLDL and apoptotic material results in activation ofautoimmunity against related antigens contributing to the development ofcardiovascular complications in SLE.

Since oxidized LDL and apoptotic material bind to the same receptors, weaimed to investigate if targeting oxidized LDL autoimmunity can affectatherosclerosis in SLE. We thus compared peripheral blood mononuclearcell (PBMC) responses to oxidized LDL in subjects with SLE and matchedhealthy controls. We also used two different approaches to modulatingimmune responses against modified LDL (mucosal tolerance induction by anapoB100 peptide linked to the B subunit of cholera toxin (CTB) andadministration of an antibody specific for the same peptide) to studythe effect of atherosclerosis development in a hypercholesterolemicmouse model of SLE.

Materials and Procedures:

SLE patients and controls: All patients in the study were evaluated by arheumatologist at the department of Rheumatology, SkånesUniversitetssjukhus, Lund. Clinical measurements were performed andfresh blood samples were collected from SLE and healthy controls during2014-2015. Peripheral blood mononuclear cells (PBMC) from 26 randomlyselected SLE individuals with age and sex matched control individualswere isolated from heparin whole blood, using Ficoll Paque (GEHealthcare) according to manufacturer's instructions. Cell numbers werecalculated and further the cells kept frozen into liquid nitrogen untilall samples were collected. Serum from each individual was collected andsaved in −80° C.

Plasma marker measurements: oxidized LDL was analysed according tomanufacturers instructions (Mercodia, Uppsala, Sweden). Plasma cytokineswere analyzed by the Proximity extension assay (PEA) technique at theClinical biomarkers facility, (Science for Life Laboratory, Uppsala,Sweden) using their 96×96 Olink Inflammation panel. Briefly,oligonucleotides-labeled antibodies in pairs were allowed to bind totheir plasma cytokine target. Upon targeting, DNA polymerase allowed foran extension and joining of the oligonucleotides creating a PCR DNAtemplate. Universal primers were added to amplify the templates to adetectable amount. Each individual DNA sequence were then detected andquantified with specific primers in a microfluid real-time quantitativePCR chip on HD Biomark instrument (96×96, Dynamic Assay IFC, FluidigmBiomark). Data analysis was performed by normalization in Olink Wizardfor GenEx (Multid Analyses, Sweden) and expressed as arbitrary units(A.U).

Flow cytometry analysis of PBMCs stimulated with DIloxLDL andMDA.P45-IgG antibody: PBMCs were slowly thawed in 37° C. PBS with 2%autologous serum, counted and seeded into appropriate medium andconditions. Medium for each stimuli condition was prepared from completeRPMI (cRPMI, 10 U/ml Penicillin/streptomycin, 1% L-glutamine, 1% sodiumpyruvate, 1% Hepes and 0.1% mercaptoethanol) with 2% autologous serum(Sigma Aldrich) including the respective stimuli conditions andpre-incubated in 37° C. for 1 h before stimulating the cells with it.Condition 1; unstimulated, condition 2; 10 μg/ml Dil-OxLDL (ThermoScientific), condition 3; 10 μg/ml Dil-OxLDL with MDA.P45-IgG.(Dil-OxLDL refers to oxidized LDL complexed with Dil dye).

At time of experiment, cells were slowly thawed in 37° C. PBS containing2% autologous serum. Cells were counted and all cells obtained weredivided into three wells for respective condition mentioned above for 24h at 37° C. in 5% CO2. After 24 h stimulation, medium was harvested andkept in −80° C. until cytokine analysis. Cells were washed twice in FACSbuffer (PBS 1% bovine serum albumin 500 mM EDTA), then stained withZombie Aqua viability dye for 20 minutes in room temperature (RT). Thiswas followed by another wash then incubation with anti-HLA-DR APC-Cy7anti-CD3-PECy7 and anti-CD14-AF700 for 30 minutes in RT prioracquisition in Gallios flow cytometer (Beckman Coulter, Brea, Calif.,USA). Cell populations were manually gated in FlowJo LTT (Tree Star, SanDiego, Calif., USA).

Multiplex analysis of PBMC cell culture medium: Cell culture medium fromhuman SLE and control PBMCs were thawed on ice before analyzed usingMultiplex Inflammation assay (Merck, Millipore, Burlington, Mass., USA)according to following kit instructions.

Mice: Female B6.lpr.ApoE−/− mice were generated and bought from JacksonLaboratories (Charles River Laboratories, Germany) before purchasingbred in our animal facility. High fat diet (HFD, 0.15% cholesterol, 21%fat, Lantmännen, Sweden) was introduced from 6 weeks of age until theexperimental end point. The ApoB100 peptide 45 (P45) was coupled tocholera toxin B (P45-CTB). Female mice were used for the treatment withp45-CTB conjugate (5 μg/injection, n=13 or CTB (30 n=13) started at 18weeks of age at day 0 and were orally administered three times the firstweek followed by once weekly until 26 weeks of age followed by sacrifice27 weeks of age. Male B6.lpr.ApoE−/− mice were used for the IgG1antibody study with administration of human recombinantmalondialdehyde-modified P45 antibody (MDA-P45-IgG1, BioInvent, Lund,Sweden). HFD was introduced at 6 weeks of age until the experimental endpoint at 22 weeks of age. Mice were treated with 1 mg MDA-P45-IgG1 (IgGthat binds to MDA-modified P45) once a week for three times with startat 18 weeks of age (n=9 in each group) followed by sacrifice at week 22.At euthanizing, urine and blood for plasma was collected before awhole-body perfusion with PBS followed by collection of heart, kidneyand carotids. Mesenterial lymph node were collected from mice treatedwith P45-CTB. All organs were snap frozen in liquid nitrogen and storedat −80° C. until further analysis. Aortas were dissected free fromsurrounding tissues and fixed in Histochoice (Amresco, Solon Ohio, USA).Spleens and mesenteric lymph nodes were collected and kept on ice untilfurther restimulation challenge in vitro.

Lipid staining of the aortic arch: The aortic arch was prepared en faceand washed in distilled water, 78% methanol and stained for 40 minutesin 0.16% Oil-Red-O dissolved in 78% methanol (0.2 mol/L NaOH). Thequantification was performed blindly using BioPix i.Q 2.0 software(Biopix AB, Gothenburg, Sweden) where bordeaux coloured regions werereferred to the content of neutral lipids in plaques.

Immunohistochemistry: Frozen hearts and kidney were embedded in TissueTek (Sakura Fine Tek., Japan) and cross sections from the aortic rootwere collected with a thickness of 8 μm and 5 μm, respectively. Kidneysections were stained in Mayers hematoxyline for 3 minutes, followed byddH2O wash twice followed by dehydration and mounting with Pertex(Sigma). For hearts sections, CD68 staining was performed using rabbitanti-CD68 (Abcam, Cambridge, UK). Briefly, sections were washed inbetween all steps with phosphate buffered saline (PBS) and Tris bufferedsaline (TBS) respectively. For permeabilization 0.5% Triton X-100 (MerckMillipore, United States) in PBS/TBS was used and further and incubatedin 3% H2O2 in PBS/TBS. Sections were blocked with 10% goat serum(Sigma-Aldrich, United States) in PBS followed by incubation with 1μg/ml of the primary anti-CD68 antibody overnight at 4° C. in a pre-wetchamber. For detection, a secondary biotinylated goat anti-rabbit IgG(Vector Labs, CA, USA) was stained with for 1 h in RT. Color wasdeveloped with DAB Detection Kit (Vector Labs) binding to thebiotinylated antibody. Heart CD68 immune stained areas and kidneyglomeruli cells and area were quantified in BioPix i.Q 2.0 software(Goteborg, Sweden).

Plasma cytokine analysis: Plasma from P45-CTB and MDA.P45-IgG treatedmice was thawed on ice for multiplex analysis of cytokines usingMilliplex Map Kit Mouse High Sensitivity T cell Magnetic Bead panel(Merck Millipore, Darmstadt, Germany) or MilliPlex (Billerica Mass.,USA) respectively, according to instructions provided. Results wereobtained by Luminex technique (Bio-Rad, CA, USA).

Splenocyte restimulation challenge in vitro: Spleens from P45-CTBtreated mice were pressed trough a 70 μM single cell mesh, washed inRPMI and centrifuged 700×g for 5 minutes. Red blood cells were lysedwith Red Blood Cell Lysis buffer (Sigma). Cells were calculated, washedin RPMI and centrifuged 700×g for 5 minutes followed by resuspension incRPMI supplemented with 2% FBS and seeding at a density of 0.5×10⁶cells/well. Cells were stimulated with CD3/28 beads (Life Technologies)and incubated for 72 h in 37° C., 5% CO2.

Plasma oxLDL, cholesterol and triglycerides assay: Plasma from all micewere thawed on ice and subsequently total plasma cholesterol andtriglyceride levels were quantified with oxLDL ELISA kit (Cloud-CloneCorp) and colorimetric assays INFINITY™ Cholesterol and Triglyceride(INT) according to the manufacturer's instructions.

Plasma anti-nuclear antibody and anti-dsDNA measurement: Plasmaanti-nuclear antibodies from P45-CTB treated mice were measured withNova-lite Hep2 slides (Inova Diagnostics, San Diego, USA). Plasma(1:100) in PBS with 0.2% BSA were incubated on the Hep2 slides 1 h atroom temperature. Slides were then washed with PBS 5 times using asqueeze bottle and put in a joplin jar filled with PBS for 5 minutestwice and then dipped 5 times in dH₂O. Slides were then incubated with25 μl human IgG (H+L)-FITC (Southern Biotech, Birmingham, USA) diluted1:100 in PBS with 0.2% BSA. Slides were then washed as previously andthereafter mounted with Vectashield (Vector Biolabs). ANA score wasassessed according to a standard matrix from four individualsindependently of each other.

Blood urea nitrogen and albumin/creatinine measurement in urine: Bloodurea nitrogen from P45-CTB was measured in urine diluted 1:100 using BUNassay (Bioassay Systems, San Francisco, USA) and urine albumin andcreatinine from all mice were measured with ELISA for albumin andcreatinine (Abcam) respectively according to manufacturer's instructionsprovided in the kits.

Statistics: Clinical comparisons from human individuals were evaluatedand statistically tested using SPSS software. For same-individualanalyses, paired nonparametric Wilxocon rank test was used. For normallydistributed data, Students t-test was used. For not normally distributeddata, Mann-Whitney rank test was used. Numerical data is expressed asmean±standard deviation if normally distributed or median±intra-quartilerange if not normally distributed. Carotid gene expressions were logtransformed and expressed as arbitrary units (A.U) and relative foldchange respectively. All data was plotted in GraphPad Prism andsignificance levels used are *=p<0.05 **=p<0.01, ***=p<0.001.

Results

The cohort consisted of a total of 51 SLE cases and 32 sex andage-matched healthy controls. Clinical characteristics of the SLEpatients are shown in Table 37.

TABLE 37 Baseline characteristics SLE individuals Lund cohort. Baselinecharacteristics SLE cases (n = 53) Age (mean) 54.3 ± 15.9 Gender (f/m %)88.7/11.3 Disease duration (median) 17.3 (10.3-25.3) SLEDAI diseasescore (mean) 2.5 ± 2.4 SLICC damage score (mean) 1.2 ± 1.4 ANA positive(%) 79.2 Corticosteroid treatm. (%) 54.7 Immunomodulating treatm. (%)62.3 Note: f; female, m; male, SLEDAI; SLE disease activity index,SLICC; Systemic Lupus Collaborating Clinics, ANA; anti-nuclear antibody.

Response of PBMCs from SLE patients and healthy controls to oxLDL:

There was no difference in the plasma level of oxLDL between SLEpatients and controls, while both interleukin (IL)-6 and monocytechemoattractant protein (MCP)-1 levels were higher in SLE patients(FIGS. 37A-37C). The plasma level of IL-6 demonstrated a significantcorrelation with oxLDL in SLE patients (r=0.32, p<0.05) but not incontrols (r=0.11, n.s.). No correlation was observed between the plasmalevels of oxLDL and MCP-1.

PBMC from 26 randomly selected SLE cases and 26 age- and sex-matchedhealthy controls were used for in vitro stimulation with oxLDL. Thefraction of CD3+ T cells was similar in SLE and control PBMC (around 50%of all viable cells), but the T cell population was considerably moreheterogeneous in the samples from SLE patients as assessed byt-Distributed Stochastic Neighbor Embedding (t-SNE), a 2-dimensionalityreduction algorithm (FIG. 38). Exposure of PBMC to 10 μg/ml of oxLDL for24 hours increased the fraction of CD3+ T cells in both groups (13.0 and16.9% respectively in controls and cases) with no significant differencebetween the two groups (FIGS. 39A, 39B and 39C). In contrast, oxLDLexposure decreased the fraction of antigen-presenting cells (APCs,defined as CD3-HLA-DR+ cells) in both control and SLE PBMC, again withno significant difference between the two groups (FIGS. 40A, 40B and40C). In view of the correlation between plasma levels of oxLDL and IL-6found in SLE patients but not in controls (FIG. 41A-41C), we nextanalyzed if oxLDL induced a larger release of IL-6 from SLE than fromcontrol PBMC. However, exposure of the cells to 10 μg/ml of oxLDL for 24hours did not affect the release of IL-6 from control or from SLE PBMC(FIG. 42A). In contrast, the release of interferon (IFN)-γ was markedlyincreased, while that of the anti-inflammatory cytokine IL-1 receptor a(IL-1RA) was reduced in both control and SLE PMBC with no significantdifference between the groups for either cytokine (FIGS. 42A-42C and43A-43C). There was no effect of oxLDL on the release of IL-10 (FIGS.44A-44C).

Effect of mucosal immunization with an ApoB100 peptide onatherosclerosis in hypercholesterolemic mice with a SLE-like phenotype:

With the exception of stronger correlation between circulating oxLDL andIL-6, the studies described above failed to identify a stronger immuneresponse against oxLDL in SLE patients. However, since the response wascomparable to that of controls and traditional approaches forcardiovascular prevention (such as statins) are less effective in SLE wedecided that it still was justified to investigate the effect of LDLimmunomodulatory therapy in a hypercholesterolemic mouse model with aSLE-like phenotype (B6.lpr.ApoE−/− mice). These mice display SLE-likecharacteristics with lymphadenopathy and splenomegaly (FIG. 45A) as aresult of Fas receptor loss (lpr) and are atherosclerosis-prone due toapolipoprotein E-deficiency (ApoE−/−). Expansion of double negative Tcells and detection of plasma anti-nuclear antibodies further confirmedthe SLE phenotype of the B6.lpr.ApoE−/− mice (FIG. 45B).

Since SLE involves an impaired ability to control for activation ofautoimmunity we first tested an immune suppressive approach. To achievethis we generated a fusion protein of a peptide sequence in ApoB100(P45; amino acids 661-680) and the cholera toxin B subunit (CTB) inwhich CTB facilitates mucosal uptake through binding to the GM1ganglioside on gut epithelial cells. B6.lpr.ApoE−/− mice were first fedhigh-fat diet for 12 weeks, and then given three oral administrations ofp45-CTB or CTB alone during one week followed by weekly administrationsfor another 8 weeks (FIG. 46). The treatment did not affect total bodyweight, plasma cholesterol or triglyceride levels (FIGS. 47A-47C). Therewas also no effect on P45 antibody levels (FIGS. 48A and 48B). Analysesof cell populations in mesenteric lymph nodes by flow cytometrydemonstrated that p45-CTB treatment was associated an increased fractionof FoxP3+/CD25+ Tregs, CD44hi/CD62− effector memory T cells andCD21/35+/CD23+/CD24− follicular B cells, while there was no effect ofthe CD1dhi/CD5+ regulatory B cell population (FIGS. 49A, 50A, 51A, 52A).There was no effect on any of these cell populations in the spleen(FIGS. 49B, 50B, 51B, 52B). These observations are in line withactivation of an immunosuppressive, regulatory response against anorally administered antigen. Analysis of the aortic arch by en face OilRed O staining demonstrated that treatment with p45-CTB was associatedwith a 32.5% decrease atherosclerosis (FIG. 53). There was no change insubvalvular plaque area (FIG. 54), but the subvalvular plaque Oil Red Oand CD68 macrophage stained area was reduced by 50.3% and 40.3%respectively in response to p45-CTB treatment (FIGS. 55 and 56). Inorder to investigate presence of immune regulatory activity in arterialtissue we analyzed FoxP3 and IL-10 mRNA levels in the carotid arteriesbut found no difference between CTB and p45-CTB treated mice (FIGS. 57Aand 57B).

To address the question whether p45-CTB treatment caused adverse effectson other SLE manifestations, we analyzed the expression of anti-nuclearantibodies (ANA) and anti-double stranded DNA antibodies (anti-dsDNA) aswell as the renal function markers albumin/creatinine ratio and bloodurea nitrogen (BUN). We observed no effect of treatment on ANA,anti-dsDNA antibodies or the albumin/creatinine ratio (ACR) (FIGS.58-60), but the mice treated with p45-CTB had significantly lower bloodurea nitrogen (BUN) levels indicating an improved renal function (FIG.61).

Effect of passive immunization with an anti-ApoB100 peptide antibody onatherosclerosis in hypercholesterolemic mice with a SLE-like phenotype:

In our second approach to influencing immune responses against oxLDL inB6.lpr.ApoE−/− mice, we applied a passive immunization strategy using arecombinant human IgG1 antibody targeting malondialdehyde (MDA)-modifiedp45 (MDA-p45 IgG). B6.lpr.ApoE−/− mice were thus fed high-fat diet for12 weeks and then given weekly intraperitoneal injections of 1 mg ofMDA-p45 or control IgG for three weeks (FIG. 62). Treatment with MDA-p45IgG reduced atherosclerosis in the aortic arch by 42.8% andCD68-macrophage subvalvular plaque staining by 30.5% and there was alsoa trend towards decreased lipid accumulation in subvalvular plaques(FIGS. 63A, 63B and 64). We observed no effect on the mRNA expression ofIL-10 or MCP-1 in carotid arteries (FIGS. 65A and 65B). Moreover, therewere no significant differences in plasma cholesterol, triglycerides oroxLDL between the two treatment groups (FIGS. 66A-66D). To search forsigns of possible renal damage by MDA-p45 IgG/oxLDL immune complexes weanalyzed glomerular area and cellularity in the kidneys but found nodifference between the groups (FIGS. 67A and 67B). There was also nodifference in the urine albumin/creatinine ratio between the groups.

Effect of MDA-p45 IgG on PBMC Isolated from SLE Patients:

Having observed the protective effect of MDA-p45 IgG on atherosclerosisdevelopment in B6.lpr.ApoE−/− mice, we next investigated how thisantibody affected the interaction of SLE PBMC with oxLDL. We incubatedthe cells with 10 μg/ml of fluorescently labelled oxLDL for 24 hourswith and without addition of 10 μg/ml MDA-p45 IgG. Addition of theantibody resulted in an increased cellular uptake of oxLDL (FIGS. 68Aand 68B). A tSNY plot of flow cytometry data revealed that oxLDLprimarily was taken up by CD14+ APC (CD3-/HLA-DR+ cells) and that theantibody increased both the expression of CD14 as well as the uptake ofoxLDL in CD14+ APC (FIGS. 69-72). Addition of the antibody had smalleffects on the cytokine release from SLE PBMC with only a modestincrease in the anti-inflammatory cytokine IL-1RA reaching statisticalsignificance (FIGS. 73A-73D).

Discussion

Accumulation and oxidation of LDL is considered a key pathogenic driverof the development of atherosclerotic plaques. LDL oxidation promotesinflammation through activation of both innate and adaptive immuneresponses. Since SLE is characterized by a dysfunctional control ofautoimmune responses, it is conceivable that an impaired ability tocontrol immune responses against oxLDL is of particular importance forthe development of cardiovascular complications in SLE. Moreover, it ishas been shown that LDL from patients with SLE is more susceptible tooxidation. Collectively, these findings suggest that targeting immuneresponses against oxLDL represents possible approach for prevention ofCVD in SLE. There is a significant unmet clinical need for suchtherapies since traditional preventive cardiovascular medication is lesseffective in SLE. In the present study, we found no difference in plasmalevels of oxLDL between patients with SLE and healthy controls and thatPBMC from SLE patients and from healthy controls reacted similarly withan increased release of INF-γ when exposed to oxLDL. However, plasmaIL-6 levels correlated more strongly with circulating levels of oxLDL inSLE patients than in controls, indicating that LDL oxidation may be morestressful to SLE patients when occurring in the body. Therefore, anembodiment of the invention provides selecting a subject having apositive correlation between plasma IL-6 level and circulating oxLDLlevel and exhibiting symptoms or having been diagnosed with SLE, andadministering to this subject an anti-P45 antibody, so as to treat,reduce the severity or likelihood of atherosclerosis in the subject.

We used mucosal immunization with the ApoB100-derived peptide P45genetically fused with CTB to stimulate regulatory responses against LDLin hypercholesterolemic mice with an SLE-like phenotype. This wasassociated with an expansion of Tregs in mesenteric lymph nodes, reducedplaque formation in the aorta and reduced presence of macrophages insubvalvular plaques. Our findings indicate the possibility that mucosaltolerance induction against LDL antigens could represent a usefulapproach to decrease cardiovascular risk in SLE. The mucosa-associatedlymphoid tissues play an important role in maintaining tolerance againstenvironmental antigens present in gut microflora and in food. It is anattractive possibility to take advantage of this to develop toleranceimmuno-therapies for autoimmune diseases. The athero-protective effectswere generally attributed to the generation of antigen-specific Tregs inthese studies. Collectively, these observations support the possibilityof developing novel therapies for prevention of CVD in SLE based onmucosal immunization with p45-CTB and similar LDL-derived antigens.Prior to Applicant's invention, there were few clinically successfulexamples of mucosal tolerance vaccines for autoimmune diseases reported.

In an alternative approach to inhibiting the development ofatherosclerosis in B6.lpr.ApoE−/− mice, we used injection of a humanrecombinant antibody specific for the MDA-modified 661-680 amino acidsin apo B. This IgG antibody binds only to oxidized LDL and not to nativeLDL, details seen in A. Schiopu, Circulation, 2004; 110:2047-2052. Thisantibody (orticumab) has previously been shown to inhibit plaquedevelopment on both ApoE−/− and human ApoB-transgene mice, as well as topotentiate plaque regression caused by cholesterol-lowering in LDLreceptor-deficient mice. In the present study we found a remarkable 50%reduction in subvalvular plaque development in response to threeinjections of the antibody. Importantly, there was also a markedreduction of inflammatory cells in the remaining plaques. There isrationale why this antibody may have a particularly beneficial effect inSLE. An impaired removal of apoptotic material through macrophagescavenger receptors is considered an important cause of activation ofautoimmunity in SLE. Since oxLDL binds to the same receptors it islikely to further impair that handling of apoptotic material in SLE. Ithas previously been shown that the MDA-p45 antibody effectively promotesuptake of ox LDL in cultured monocytes isolated from healthy blooddonors. In the present study, we found that this is true also for CD14+monocytes isolated from patients with SLE. Accordingly, by reroutinguptake of oxLDL from scavenger to Fc receptors the antibody may help toincrease the capacity for removal of apoptotic material by scavengerreceptors. The mechanisms through which this antibody protects againstatherosclerosis are only partly known. One likely mechanism involves thefacilitation of removal of toxic oxLDL particles from the extracellularspace which at the same time would make the cholesterol available forreverse cholesterol through adenosine triphosphate-binding cassettetransporter A1-mediated transfer to HDL. When complexed with oxLDL theantibody has also been shown to suppressive the activity of monocytes bybinding to inhibitory FcγIIb receptor. One advantage of the MDA-p45antibody is that it already has been proven to be safe in clinicalstudies. In this study, however, the antibody failed to reduce carotidplaque inflammation as assessed by 18F-fluorodeoxyglucose positronemission tomography imaging in stable cardiovascular patients. Thereason for the lack of effect of antibody-treatment in this studyremains a matter of controversy as the patients may have been in a toostable state of disease to allow identification of a response totreatment. It could also seem counter-intuitive to treat SLE patientswith an antibody against a self-antigen since such antibodies areconsidered a hallmark of the disease. Although there are experimentalevidence that some of these antibodies are pathogenic, it remains to befully elucidated if they are a cause or a consequence of the disease. Inconclusion, the present study provides experimental and clinical supportfor targeting oxLDL autoimmunity as a possible target for prevention ofcardiovascular complications in SLE.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

It is to be understood that the embodiments of the application disclosedherein are illustrative of the principles of the embodiments of theapplication. Other modifications that can be employed can be within thescope of the application. Thus, by way of example, but not oflimitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention.

The invention claimed is:
 1. A method for treating systemic lupuserythematosus (SLE) in a subject in need thereof by passiveimmunization, comprising administering a therapeutically orprophylactically effective amount of an antibody that binds at least oneoxidized fragment of apolipoprotein B100 (ApoB100), so as to treat SLE,wherein the antibody comprises a variable heavy region (V_(H)) and avariable light region (V_(L)), wherein the V_(H) region comprises thesequence set forth in SEQ ID NO: 324 and the variable light region(V_(L)) comprises the sequence set forth in SEQ ID NO:
 325. 2. Themethod of claim 1, wherein the antibody is a human antibody.
 3. Themethod of claim 1, wherein the V_(H) region consists of the sequence setforth in SEQ ID NO: 324 and the variable light region (V_(L)) consistsof the sequence set forth in SEQ ID NO:
 325. 4. A method for treating,reducing the severity of, slowing progression of or inhibiting systemiclupus erythematosus (SLE) in a subject in need thereof, comprising:administering to the subject an effective amount of an antibody orantibody fragment, wherein the antibody or antibody fragment comprisesheavy chain complementarity determining region (HCDR) 1 (HCDR1), HCDR 2(HCDR2) and HCDR 3 (HCDR3) whose sequences comprise SEQ ID NOs: 318, 319and 320, respectively, and light chain complementarity determiningregion (LCDR) 1 (LCDR1), LCDR 2 (LCDR2) and LCDR 3 (LCDR3) whosesequences comprises SEQ ID NOs: 321, 322 and 323, respectively.
 5. Themethod of claim 4, wherein the antibody or antibody fragment is capableof binding to a fragment of apolipoprotein B100 (ApoB100), and thefragment of ApoB100 comprises an amino acid sequence of SEQ ID NO:45 andis an aldehyde derivative.
 6. The method of claim 4, wherein theantibody or antibody fragment comprises a variable heavy region (V_(H))of SEQ ID NO: 324, a variable light region (V_(L)) of SEQ ID NO: 325, orboth.
 7. The method of claim 6, wherein the antibody or antibodyfragment comprises a heavy chain of SEQ ID NO: 316, a light chain of SEQID NO: 317, or both.
 8. The method of claim 4, wherein the antibody isorticumab and is administered intravenously at an initial dose of atleast 5 mg/kg, followed by a plurality of subsequent doses, each atleast 2 mg/kg/week, at least 2.5 mg/kg/two weeks, or at least 6mg/kg/month.
 9. The method of claim 8, wherein the subsequent doses areadministered over at least 2 weeks, 3 weeks, or 1 month.
 10. The methodof claim 4, wherein the subject is a human, the antibody is orticumab,and the orticumab is administered subcutaneously at a dose of about 330mg/month for about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months.
 11. Themethod of claim 4, wherein the antibody or antibody fragment isadministered at 10-150 μg/kg, 150-750 μg/kg, 1-5 mg/kg, 5-10 mg/kg,10-15 mg/kg, 15-20 mg/kg, 20-25 mg/kg, 25-50 mg/kg or 50-75 mg/kg. 12.The method of claim 4, further comprising selecting a subject exhibitingsymptoms of SLE or a subject who has been diagnosed with SLE.
 13. Themethod of claim 4, comprising administering an anti-malarialtherapeutic, a corticosteroid, mycophenolate mofetil, azathioprine,cyclophosphamide, cyclosporine, leflunomide, or methotrexate incombination with the antibody or antibody fragment to the subject.
 14. Amethod for treating, reducing the severity of, slowing progression of orinhibiting atherosclerosis in a subject exhibiting symptoms of or havingbeen diagnosed with systemic lupus erythematosus (SLE), comprising:administering to the subject an effective amount of an antibody orantibody fragment capable of binding to a fragment of apolipoproteinB100 (ApoB100), wherein the fragment of ApoB100 comprises an amino acidsequence of SEQ ID NO: 45 or an active site thereof, and wherein theantibody or antibody fragment comprises heavy chain complementaritydetermining region (HCDR) 1 (HCDR1), HCDR 2 (HCDR2) and HCDR 3 (HCDR3)whose sequences comprise SEQ ID NOs: 318, 319 and 320, respectively, andone, two or three light chain complementarity determining region (LCDR)1 (LCDR1), LCDR 2 (LCDR2) and LCDR 3 (LCDR3) whose sequences compriseSEQ ID NOs: 321, 322 and 323, respectively.
 15. The method of claim 14,wherein the antibody or antibody fragment comprises a variable heavyregion (V_(H)) of SEQ ID NO: 324, a variable light region (V_(L)) of SEQID NO: 325, or both.
 16. The method of claim 14, wherein the antibody orantibody fragment comprises a heavy chain of SEQ ID NO: 316, a lightchain of SEQ ID NO: 317, or both.
 17. The method of claim 14, whereinthe subject exhibits symptoms of atherosclerosis and SLE before theadministration, and after the administration the subject has reducedplaque volume in artery.
 18. The method of claim 14, wherein theantibody is orticumab and is administered intravenously at an initialdose of at least 5 mg/kg, followed by a plurality of subsequent doses,each at least 2 mg/kg/week, at least 2.5 mg/kg/two weeks, or at least 6mg/kg/month.
 19. The method of claim 14, wherein the subject is a human,the antibody is orticumab, and the orticumab is administeredsubcutaneously at a dose of about 330 mg/month for about 3, 4, 5, 6, 7,8, 9, 10, 11 or 12 months.
 20. The method of claim 14, wherein theatherosclerosis is accelerated atherosclerosis.